Mitapivat therapy and modulators of cytochrome p450

ABSTRACT

Described herein are methods of treating pyruvate kinase deficiency (PKD), sickle cell disease or thalassemia with mitapivat or a pharmaceutically acceptable salt thereof, or use of the drug for the treatment of these conditions, in combination with or in the absence of with a secondary drug, such as an inducer or an inhibitor of cytochrome P450. Various doses and dosing regimens of mitapivat in monotherapy and in concomitant medications are described.

RELATED APPLICATIONS

The application is a continuing application of U.S. patent applicationSer. No. 16/900,610, filed Jun. 12, 2020, which claims the benefit ofU.S. Provisional Application Ser. No. 62/861,600, filed Jun. 14, 2019,the entire teachings of which are incorporated herein by reference.

BACKGROUND

Pyruvate kinase deficiency (PKD) is a disease of the red blood cellscaused by a deficiency of the pyruvate kinase R (PKR) enzyme due torecessive mutations of PKLR gene. PKR activators can be beneficial totreat PKD, thalassemia (e.g., beta-thalassemia), abetalipoproteinemia orBassen-Kornzweig syndrome, sickle cell disease, paroxysmal nocturnalhemoglobinuria, anemia (e.g., congenital anemias) (e.g., enzymopathies),hemolytic anemia (e.g., hereditary and/or congenital hemolytic anemia,acquired hemolytic anemia, chronic hemolytic anemia caused byphosphoglycerate kinase deficiency, anemia of chronic diseases,non-spherocytic hemolytic anemia or hereditary spherocytosis). Treatmentof PKD is supportive, including blood transfusions, splenectomy,chelation therapy to address iron overload, and/or interventions forother disease-related morbidity. Currently, however, there is noapproved medicine that treats the underlying cause of PKD, and thus theetiology of life-long hemolytic anemia.

Mitapivat, also known as AG-348 or by its chemical nameN-(4-(4-(cyclopropylmethyl)piperazine-1-carbonyl)phenyl)quinoline-8-sulfonamide,and is represented by the following structural formula:

is an allosteric activator of pyruvate kinase R (PKR). See, e.g.,International Patent Application Publication Nos. WO 2011/002817 and WO2016/201227.

Mitapivat sulfate is represented by the following structural formula:

and is currently in Phase 2 and Phase 3 clinical trials as adisease-altering therapy in patients with PKD, hemolytic anemia andthalassemia. See, e.g., U.S. Clinical Trials Identifier Nos.NCT03692052, NCT03559699, NCT03548220, NCT03853798, and NCT02476916.

SUMMARY

Non-clinical studies described in Example 1 show that the PKR activator,mitapivat, is primarily metabolized by the cytochrome P450 3A4 (CYP3A4)or 3A5 (CYP3A5) enzymes (i.e., >90%), with minor contributions fromother detoxification enzymes, namely CYP2C8, CYP2C9, and CYP1A2. It hasalso been found that in a clinical trial, total exposure of mitapivatincreased in the presence of itraconazole, a strong CYP3A4A and p-gpinhibitor, compared with dosing of mitapivat sulfate alone. It hasfurther been found that total exposure of mitapivat decreased in thepresence of rifampin, a strong CYP3A4A and p-gp inducer, compared withdosing of mitapivat sulfate alone.

Accordingly, the present disclosure provides methods of treating adisease, disorder or a condition with mitapivat or a pharmaceuticallyacceptable salt thereof and a CYP3A4/5 inducer or a CYP3A4/5 inhibitor.In another aspect, the present disclosure provides methods of treating adisease, disorder or a condition with mitapivat or a pharmaceuticallyacceptable salt thereof and a p-glycoprotein (p-gp) inhibitor. As usedherein, “CYP3A4/5” means cytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).In some embodiments, the pharmaceutically acceptable salt is mitapivatsulfate.

Specifically, in one embodiment, the present disclosure relates to amethod of treating pyruvate kinase deficiency (PKD) in a subject,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof and an effective amount ofan inhibitor of cytochrome P450 3A4/5 (CYP3A4/5). In another embodiment,the present disclosure relates to a method of treating sickle celldisease in a subject, comprising administering to the subject aneffective amount of mitapivat or a pharmaceutically acceptable saltthereof and an effective amount of an inhibitor of cytochrome P450 3A4/5(CYP3A4/5). In another embodiment, the present disclosure relates to amethod of treating thalassemia (e.g., alpha-thalassemia andbeta-thalassemia) in a subject, comprising administering to the subjectan effective amount of mitapivat or a pharmaceutically acceptable saltthereof and an effective amount of an inhibitor of cytochrome P450 3A4/5(CYP3A4/5). In another embodiment, the present disclosure relates to amethod of treating hemolytic anemia in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof and an effective amount of aninhibitor of cytochrome P450 3A4/5 (CYP3A4/5). In some embodiments, theinhibitor of CYP3A4/5 is a moderate CYP3A4/5 inhibitor. In someembodiments, the inhibitor of CYP3A4/5 is a mild CYP3A4/5 inhibitor.

In another embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof and an effective amount of aninducer of cytochrome P450 3A4/5 (CYP3A4/5). In another embodiment, thepresent disclosure relates to a method of treating sickle cell diseasein a subject, comprising administering to the subject an effectiveamount of mitapivat or a pharmaceutically acceptable salt thereof and aneffective amount of an inducer of cytochrome P450 3A4/5 (CYP3A4/5). Inanother embodiment, the present disclosure relates to a method oftreating thalassemia (e.g., alpha-thalassemia and/or beta-thalassemia)in a subject, comprising administering to the subject an effectiveamount of mitapivat or a pharmaceutically acceptable salt thereof and aneffective amount of an inducer of cytochrome P450 3A4/5 (CYP3A4/5). Inanother embodiment, the present disclosure relates to a method oftreating hemolytic anemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof and an effective amount of an inducer ofcytochrome P450 3A4/5 (CYP3A4/5). In some embodiments, the inducer ofCYP3A4/5 is a moderate CYP3A4/5 inducer. In some embodiments, theinhibitor of CYP3A4/5 is a mild CYP3A4/5 inducer.

Specifically, in one embodiment, the present disclosure relates to amethod of treating pyruvate kinase deficiency (PKD) in a subject,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof and an effective amount ofa p-glycoprotein inhibitor. In another embodiment, the presentdisclosure relates to a method of treating sickle cell disease in asubject, comprising administering to the subject an effective amount ofmitapivat or a pharmaceutically acceptable salt thereof and an effectiveamount of a p-glycoprotein inhibitor. In another embodiment, the presentdisclosure relates to a method of treating thalassemia (e.g.,alpha-thalassemia and beta-thalassemia) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof and an effective amount of ap-glycoprotein inhibitor. In another embodiment, the present disclosurerelates to a method of treating hemolytic anemia in a subject,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof and an effective amount ofa p-glycoprotein inhibitor. In some embodiments, the p-glycoproteininhibitor is a moderate p-glycoprotein inhibitor. In some embodiments,the p-glycoprotein inhibitor is a mild p-glycoprotein inhibitor.

The present disclosure further provides methods of treating a disease,disorder or a condition with mitapivat or a pharmaceutically acceptablesalt thereof in the absence of a CYP3A4/5 inducer or a CYP3A4/5inhibitor or p-glycoprotein inhibitor.

Specifically, in one embodiment, the present disclosure relates to amethod of treating pyruvate kinase deficiency (PKD) in a subject,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof in the absence of aninducer or an inhibitor of cytochrome P450 3A4/5 (CYP3A4/5),respectively. In another embodiment, the present disclosure relates to amethod of treating sickle cell disease in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of an inducer oran inhibitor of cytochrome P450 3A4/5 (CYP3A4/5), respectively. In yetanother embodiment, the present disclosure relates to a method oftreating thalassemia (e.g., alpha-thalassemia and/or beta-thalassemia)in a subject, comprising administering to the subject an effectiveamount of mitapivat or a pharmaceutically acceptable salt thereof in theabsence of an inducer or an inhibitor of cytochrome P450 3A4/5(CYP3A4/5), respectively. In yet another embodiment, the presentdisclosure relates to a method of treating hemolytic anemia in asubject, comprising administering to the subject an effective amount ofmitapivat or a pharmaceutically acceptable salt thereof in the absenceof an inducer or an inhibitor of cytochrome P450 3A4/5 (CYP3A4/5),respectively. In some embodiments, the inducer of CYP3A4/5 is a stronginducer of CYP3A4/5. In some embodiments, the inducer of CYP3A4/5 is amoderate inducer of CYP3A4/5. In some embodiments, the inhibitor ofCYP3A4/5 is a strong inhibitor of CYP3A4/5. In some embodiments, theinhibitor of CYP3A4/5 is a moderate inhibitor of CYP3A4/5.

In another embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of ap-glycoprotein inhibitor. In another embodiment, the present disclosurerelates to a method of treating sickle cell disease in a subject,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof in the absence of ap-glycoprotein inhibitor. In yet another embodiment, the presentdisclosure relates to a method of treating thalassemia (e.g.,alpha-thalassemia and/or beta-thalassemia) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of ap-glycoprotein inhibitor. In yet another embodiment, the presentdisclosure relates to a method of treating hemolytic anemia in asubject, comprising administering to the subject an effective amount ofmitapivat or a pharmaceutically acceptable salt thereof in the absenceof a p-glycoprotein inhibitor. In some embodiments, the p-glycoproteininhibitor is a strong p-glycoprotein inhibitor. In some embodiments, thep-glycoprotein inhibitor is a mild p-glycoprotein inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing Arithmetic Mean (SD) Plasma Mitapivat andMetabolite Concentration-Time Profiles following administration of 20 mgmitapivat sulfate alone (Treatment Period 1) and co-administered withmultiple doses of 200 mg itraconazole (Treatment Period 2) on linearscale (Part 1). “Metabolite” refers to the CYP3A4 metabolite ofmitapivat (see Example 2).

FIG. 2 is a graph showing Arithmetic Mean Plasma Mitapivat andMetabolite Concentration-Time Profiles following administration of 20 mgmitapivat sulfate alone (Treatment Period 1) and co-administered withmultiple doses of 200 mg itraconazole (Treatment Period 2) on semi-logscale (Part 1) (see Example 2).

FIG. 3 is a graph showing Arithmetic Mean (SD) Plasma Mitapivat andMetabolite Concentration-Time Profiles following administration of 50 mgmitapivat sulfate alone (Treatment Period 1) and co-administered withmultiple doses of 600 mg rifampin on linear scale (Treatment Period 2,Part 2, see Example 2).

FIG. 4 is a graph showing Arithmetic Mean Plasma Mitapivat andMetabolite Concentration-Time Profiles following administration of 50 mgmitapivat sulfate alone (Treatment Period 1) and co-administered withmultiple doses of 600 mg rifampin on semi-log scale (Treatment Period 2,Part 2, see Example 2).

DETAILED DESCRIPTION Definitions

A “CYP3A4/5 modulator” refers to any substance, including, but notlimited to a small molecule (with a molecular weight of ≤1000 Da, or≤750 Da, or ≤500 Da), a nucleoside, a nucleotide, a nucleobase, a sugar,a nucleic acid, an amino acid, a polysaccharide, a peptide, apolypeptide, a protein, an antibody, an aptamer, a natural or syntheticplant-based substance, or a complex or a composition comprising anycombination of the aforementioned substances, that can modify or alterthe activity and/or expression levels of the enzyme cytochrome P450 3A4(CYP3A4; EC 1.14.13.97) or 3A5 (CYP3A5; EC1.14.14.1). Briefly, thecytochrome P450 (CYP) is a superfamily of detoxification enzymes thatare responsible for the oxidative and reductive metabolic transformationof drug medications and the ultimate “clearance”, which is theelimination of the drug from the body as determined by the measuredplasma levels of the drug and optionally, the drug metabolites, such asthe metabolite known to be formed by CYP3A4/5. CYP3A4 and CYP3A5 are twoof such enzymes and are largely prevalent in the liver.

A CYP3A4/5 modulator may be a CYP3A4/5 inducer or a CYP3A4/5 inhibitor.A “CYP3A4/5 inducer” or “inducer of CYP3A4/5” is a CYP3A4/5 modulator asdefined above that can increase, enhance, stimulate, induce, accelerateor augment the CYP3A4/5 activity from its existing state or nativestate, such as by at least about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% and 100% or more relative to a reference level,e.g., the level of the CYP3A4/5 activity and/or expression prior to theexposure or contact with the CYP3A4/5 inducer, including all ranges andvalues therebetween not expressly mentioned above. The CYP3A4/5 induceractivity may cause mitapivat or a pharmaceutically acceptable saltthereof that is co-administered with the CYP3A4/5 inducer to bemetabolized and eliminated more quickly from the body, as determined bythe measured plasma levels of the compound and its metabolites.

A “CYP3A4/5 inhibitor” or “inhibitor of CYP3A4/5” is a CYP3A4/5modulator as defined above that can decrease, reduce, inhibit,decelerate, attenuate, or suppress the CYP3A4/5 activity and/orexpression from its existing state or native state, such as by at leastabout 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% and100% or more relative to a reference level, e.g., the level of theCYP3A4/5 activity and/or expression prior to the exposure or contactwith the CYP3A4/5 inhibitor, including all ranges and valuestherebetween not expressly mentioned above. The CYP3A4/5 inhibitor maycause mitapivat or a pharmaceutically acceptable salt thereof that isco-administered with the CYP3A4/5 inhibitor to be metabolized andeliminated more slowly from the body, as determined by the measuredplasma levels of the compound and its metabolites.

If an investigational drug is a CYP inducer, it can be classified as astrong, moderate, or mild inducer based on its effect on an index CYPsubstrate. The convention is to categorize CYP induction in thefollowing ways:

A strong inducer decreases the AUC of a sensitive index CYP by ≥80percent.

A moderate inducer decreases the AUC of a sensitive index CYP substrateby ≥50 to <80 percent.

A mild inducer decreases the AUC of a sensitive index CYP substrate by≥20 to <50 percent.

A CYP inhibitor can be classified as a strong, moderate, or mildinhibitor based on its effect on an index CYP substrate. A stronginhibitor increases the AUC of a sensitive index CYP substrate ≥5-fold.A moderate inhibitor increases the AUC of a sensitive index CYPsubstrate by ≥2- to <5-fold. A mild inhibitor increases the AUC of asensitive index CYP substrate by ≥1.25- to <2-fold.

A p-glycoprotein inhibitor refers to any substance, including, but notlimited to a small molecule (with a molecular weight of ≤1000 Da, or≤750 Da, or ≤500 Da), a nucleoside, a nucleotide, a nucleobase, a sugar,a nucleic acid, an amino acid, a polysaccharide, a peptide, apolypeptide, a protein, an antibody, an aptamer, a natural or syntheticplant-based substance, or a complex or a composition comprising anycombination of the aforementioned substances, that can decrease, reduce,inhibit, decelerate, attenuate, or suppress the p-glycoprotein activityand/or expression from its existing state or native state, such as by atleast about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99% and 100% or more relative to a reference level, e.g., the level ofthe p-glycoprotein. In some embodiments, the p-glycoprotein inhibitor isa strong p-glycoprotein inhibitor, selected from the group consisting ofamiodarone, azithromycin, captopril, carvedilol, clarithromycin,conivaptan, cyclosporine, diltiazem, dronedarone, erythromycin,felodipine, itraconazole, ketoconazole, lopinavir, ritonavir, quercetin,quinidine, ranolazine ticagrelor, and erapamil.

The terms “effective amount” and “therapeutically effective amount” areused interchangeably throughout the present disclosure, whetherreferring to mitapivat or a pharmaceutically acceptable salt thereof, aCYP3A4/5 inducer, or a CYP3A4/5 inhibitor.

An “effective amount” of mitapivat or a pharmaceutically acceptable saltthereof is an amount sufficient to provide a therapeutic benefit in thetreatment of pyruvate kinase deficiency (PKD), thalassemia (e.g.,alfa-thalassemia, beta-thalassemia or non-transfusion-dependentthalassemia), abetalipoproteinemia or Bassen-Kornzweig syndrome, sicklecell disease, paroxysmal nocturnal hemoglobinuria, anemia (e.g.,congenital anemias (e.g., enzymopathies), hemolytic anemia (e.g.,hereditary and/or congenital hemolytic anemia, acquired hemolyticanemia, chronic hemolytic anemia, chronic hemolytic anemia caused byphosphoglycerate kinase deficiency, anemia of chronic diseases,non-spherocytic hemolytic anemia or hereditary spherocytosis), as wellas all other conditions disclosed in the section “Diseases, Disordersand Conditions” in the present disclosure, i.e., conditions that areresponsive to treatment with a PKR activator such as mitapivat or apharmaceutically acceptable salt thereof. Such diseases, disorders andconditions are hereinafter referred as “a PKR activator-responsivecondition” when referring to any one of these conditions or “PKRactivator-responsive conditions” when referring to these conditionscollectively. Additionally or alternatively, an “effective amount” ofmitapivat or a pharmaceutically acceptable salt thereof is an amountsufficient to delay or minimize one or more effects or symptomsassociated with these conditions. In one aspect, an “effective amount”of mitapivat or a pharmaceutically acceptable salt thereof means anamount of mitapivat or a pharmaceutically acceptable salt thereof, aloneor in combination with other therapies, which provides a therapeuticbenefit in the treatment of the condition. The term “effective amount”can encompass an amount that improves overall therapy, reduces or avoidseffects, symptoms, signs, or causes of the condition, and/or enhancesthe therapeutic efficacy of another therapeutic agent. In certainembodiments, an “effective amount” of mitapivat or a pharmaceuticallyacceptable salt thereof is an amount sufficient for eliciting measurableactivation of wild-type or mutant PKR. In certain embodiments, an“effective amount” of mitapivat or a pharmaceutically acceptable saltthereof is an amount sufficient for regulating 2,3-diphosphoglyceratelevels in blood in need thereof or for treating PKR activator-responsiveconditions or treating diseases or conditions that are associated withincreased 2,3-diphosphoglycerate levels (e.g., liver diseases). Incertain embodiments, an effective amount of mitapivat or apharmaceutically acceptable salt thereof is an amount sufficient foreliciting measurable activation of wild-type or mutant PKR and forregulating 2,3-diphosphoglycerate levels in blood in need thereof or fortreating PKR activator-responsive conditions or treating diseases orconditions that are associated with increased 2,3-diphosphoglyceratelevels (e.g., liver diseases). In one aspect, the effective amount isthe amount required to generate a subject's hemoglobin response of ≥1.0g/dL (such as ≥1.5 g/dL or ≥2.0 g/dL) increase in Hb concentration frombaseline. In one aspect, the subject's baseline Hb concentration is theaverage of all available Hb concentrations before treatment with acompound described herein. In certain aspects, the effective amount isthe amount required to reduce the patient's transfusion burden. In oneaspect, the effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) is between about0.01-100 mg/kg body weight/day of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate), e.g., about 0.1-100mg/kg body weight/day.

Concomitant medications will be commonly used with mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate). An“effective amount” of the drug (e.g., an effective amount of a CYP3A4/5inducer or an effective amount of a CYP3A4/5 inhibitor) beingco-administered with mitapivat or a pharmaceutically acceptable saltthereof (e.g., mitapivat sulfate) is an amount sufficient to provide adesired therapeutic benefit. Drugs that are being co-administered withmitapivat or a pharmaceutically acceptable salt thereof (e.g., mitapivatsulfate), including some of the current antiarrhythmics, antibiotics,antidepressants, antifungals, calcium channel blockers, H₂ receptorantagonists, non-nucleoside reverse transcriptase inhibitors (NNRTI),protease inhibitors, etc., will sometimes be CYP3A4/5 inducers orCYP3A4/5 inhibitors. In such instances, the amount of mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate) orthe amount of the drug being co-administered should be adjusted toreduce effects of a drug-drug interaction between mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate) andthe co-administered drug.

In one aspect, an effective amount of a CYP3A4/5 inducer is betweenabout 0.1-1000 mg/kg/day of a CYP3A4/5 inducer, e.g., about 1.0-1000mg/kg/day, depending on the drug that is co-administered with.

In one aspect, an effective amount of a CYP3A4/5 inhibitor is betweenabout 0.1-1000 mg/kg/day of a CYP3A4/5 inhibitor, e.g., about 1.0-1000mg/kg/day, depending on the drug that is co-administered with.

In one aspect, an effective amount of a p-gp inhibitor is between about0.1-1000 mg/kg/day of a p-gp inhibitor, e.g., about 1.0-1000 mg/kg/day,depending on the drug that is co-administered with.

As used herein, reduction in transfusion burden means at least 20%reduction in the number of RBC units transfused within at least 5 weeksof treatment. In certain embodiments, the reduction in transfusionburden is ≥33% reduction in the number of RBC units transfused within atleast 5 weeks of treatment. In certain embodiments, reduction oftransfusion burden is ≥33% reduction in the number of RBC unitstransfused within at least weeks (e.g., at least 20 weeks or at least 24weeks) of treatment.

The term “activating” as used herein means an agent that (measurably)increases the activity of wild type pyruvate kinase R (wt PKR) or causeswild type pyruvate kinase R (wt PKR) activity to increase to a levelthat is greater than wt PKR's basal levels of activity or an agent that(measurably) increases the activity of a mutant pyruvate kinase R (mPKR)or causes mutant pyruvate kinase R (mPKR) activity to increase to alevel that is greater than that mutant PKR's basal levels of activity,for examples, to a level that is 20%, 40%, 50%, 60%, 70%, 80%, 90% or100% of the activity of wild type PKR.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of a disease or disorder, or one or more effects or symptomsthereof, as described herein. In some embodiments, treatment may beadministered after one or more symptoms have developed, i.e.,therapeutic treatment. In other embodiments, treatment may beadministered in the absence of symptoms. For example, treatment may beadministered to a susceptible individual prior to the onset of symptoms(e.g., in light of a history of symptoms and/or in light of genetic orother susceptibility factors), i.e., prophylactic treatment. Treatmentmay also be continued after symptoms have resolved, for example toreduce the likelihood of or delay their recurrence.

As used herein the terms “subject” and “patient” may be usedinterchangeably and means a mammal in need of treatment with mitapivator a pharmaceutically acceptable salt thereof (e.g., mitapivat sulfate).

The term “pharmaceutically acceptable salt” when referring to apharmaceutically acceptable salt of mitapivat, refers to those saltswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and lower animals without unduetoxicity, irritation, allergic response, and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art, for example, Berge et al.describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19. Pharmaceutically acceptablesalts of mitapivat include those derived from suitable inorganic andorganic acids. Examples of pharmaceutically acceptable acid additionsalts are salts of an amino group formed with inorganic acids, such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids, such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, besylate bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, gentisate, hemisulfate,heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, tosylate p-toluenesulfonate,undecanoate, valerate salts, and the like. Preferably, apharmaceutically acceptable salt of mitapivat is a sulfate salt.

As used herein, the terms “about” and “approximately” are used herein tomean within the typical ranges of tolerance in the art. In oneembodiment, “about” means within 2 standard deviations from the meanvalue. In one embodiment, “about” means ±10%. In one embodiment, “about”means ±5%. When “about” is present before a series of numbers of numberranges, it is understood that the term can apply to any and each of thenumbers and ranges recited in the series.

Compositions and Administration

The effective amount of mitapivat, in accordance with a method of theinvention, may be administered in the form of a pharmaceuticalcomposition comprising mitapivat or a pharmaceutically acceptable saltthereof (e.g., mitapivat sulfate), together with a pharmaceuticallyacceptable carrier.

Pharmaceutical compositions of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) into association witha carrier and/or one or more other accessory ingredients, and then, ifnecessary and/or desirable, shaping and/or packaging the product into adesired single- or multi-dose unit.

Pharmaceutically acceptable carriers used in the manufacture of providedpharmaceutical compositions include inert diluents, dispersing and/orgranulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Exemplary carriers, diluents,granulating and/or dispersing agents, surface active agents and/oremulsifiers, binding agents, preservatives, antioxidants, chelatingagent, antifungal preservatives, alcohol preservatives, acidicpreservatives, other types of preservatives, buffering agents,lubricating agents, and natural oils that can be included in apharmaceutical composition of mitapivat or a pharmaceutically acceptablesalt thereof (e.g., mitapivat sulfate) as disclosed in InternationalPatent Application No. WO2019/104134.

Pharmaceutical compositions of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, transmucosally, or in an ophthalmic preparation. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. In one aspect, the pharmaceutical compositionsprovided herewith are orally administered in an orally acceptable dosageform including, but not limited to, capsules, tablets, emulsions andaqueous suspensions, dispersions and solutions. In the case of tabletsfor oral use, carriers which are commonly used include one or moresubstances selected from microcrystalline cellulose, mannitol,croscarmellose sodium, and sodium stearyl fumarate. Lubricating agents,such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

A secondary drug can be co-administered with mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate). Theeffective amount of a secondary drug that is co-administered withmitapivat or a pharmaceutically acceptable salt thereof (e.g., mitapivatsulfate), such as a CYP3A4/5 inducer or a CYP3A4/5 inhibitor or a p-gpinhibitor, if used in a method of the invention, may also beadministered in the form of a pharmaceutical composition comprising theCYP3A4/5 inducer or the CYP3A4/5 inhibitor or the p-gp inhibitor,together with a pharmaceutically acceptable carrier. Pharmaceuticalcompositions of the secondary drug may be prepared and administered asdescribed above for pharmaceutical compositions of mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate).

In one aspect, mitapivat or a pharmaceutically acceptable salt thereof(e.g., mitapivat sulfate) is formulated as a tablet composition togetherwith a pharmaceutically acceptable carrier, in accordance with thedisclosures of International Patent Application No. WO 2019/104134.

In one embodiment, in therapies comprising mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate) and asecondary drug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor),mitapivat or a pharmaceutically acceptable salt thereof (e.g., mitapivatsulfate) and the secondary drug are administered “concurrently” to thesubject, which means that the subject is administered both drugs on thesame day on all days of the treatment period. In one embodiment,mitapivat or a pharmaceutically acceptable salt thereof (e.g., mitapivatsulfate) and the secondary drug are administered “sequentially” to thesubject, which means that the subject is administered either of the twodrugs on all days of the treatment period, but not both drugs. Inanother embodiment, mitapivat or a pharmaceutically acceptable saltthereof (e.g., mitapivat sulfate) and the secondary drug areadministered “sequentially and concurrently” to the subject, which meansthat the subject is administered either of the two drugs on certain daysof the treatment period, but not both drugs; and the subject isadministered both drugs on the same day on other days of the treatmentperiod.

Doses and Dosing Regimens

The amount of mitapivat or a pharmaceutically acceptable salt thereof(e.g., mitapivat sulfate) that may be combined with carrier materials toproduce a composition in a single dosage form will vary depending uponthe subject to be treated and the particular mode of administration. Forexample, a specific dosage and treatment regimen for any particularsubject will depend upon a variety of factors, including age, bodyweight, general health, sex, diet, time of administration, rate ofexcretion, drug combination, the judgment of the treating physician, andthe severity of the particular disease being treated. The amount ofmitapivat or a pharmaceutically acceptable salt thereof will also dependupon the particular form (e.g., free base, salt form, crystalline form,etc.) in the composition. In one aspect, a provided composition may beformulated such that a dosage equivalent to about 0.001 to about 100mg/kg body weight/day of mitapivat or a pharmaceutically acceptable saltthereof (e.g., about 0.5 to about 100 mg/kg of mitapivat or apharmaceutically acceptable salt thereof) can be administered to asubject receiving these compositions. Alternatively, dosages equivalentto 1 mg/kg and 1000 mg/kg of mitapivat or a pharmaceutically acceptablesalt thereof every 4 to 120 hours is also acceptable.

In one aspect, mitapivat or a pharmaceutically acceptable salt thereofis formulated for administration at a dose of equivalent to about 2 mgto about 3000 mg of mitapivat. In certain embodiments, the dose is anoral dose. In certain embodiments, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated equivalent to about 2 mg to about3000 mg of mitapivat. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated equivalent toabout 5 mg to about 350 mg of mitapivat. In certain embodiments,mitapivat or a pharmaceutically acceptable salt thereof is formulatedequivalent to about 5 mg to about 200 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 5 mg to about 100 mg of mitapivat. Incertain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated equivalent to about 5 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 10 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 15 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 20 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 25 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 30 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 35 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 40 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 45 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 50 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 55 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 60 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 65 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 70 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 75 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 80 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 85 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 90 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 95 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 100 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 110 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 120 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 130 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 140 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 150 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 160 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 170 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 180 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 190 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 200 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 210 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 220 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 230 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 240 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 250 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 260 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 270 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 280 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 290 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 300 mg of mitapivat. In certainembodiments, mitapivat or a pharmaceutically acceptable salt thereof isformulated equivalent to about 300 to 360 mg of mitapivat. In certainembodiment, the pharmaceutically acceptable salt of mitapivat ismitapivat sulfate.

In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about 2mg to about 3000 mg of mitapivat per day. In certain embodiments,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose equivalent to about 5 mg to about 500 mg ofmitapivat of per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 5 mg to about 200 mg ofmitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 5 mg of mitapivat per day.In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about 5mg to about 10 mg of mitapivat per day. In certain embodiments,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose of about 15 mg equivalent to mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 20 mg of mitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 25 mg of mitapivat per day.In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about30 mg of mitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 35 mg of mitapivat per day.In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about40 mg of mitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 45 mg of mitapivat per day.In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about50 mg of mitapivat thereof per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 55 mg of mitapivat thereofper day. In certain embodiments, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 60 mg of mitapivat per day. In certain embodiments,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose equivalent to about 70 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 80 mg of mitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 90 mg of mitapivat per day.In certain embodiments, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about100 mg of mitapivat per day. In certain embodiments, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 110 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 120 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 130 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 140 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 150 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 160 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 170 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 180 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 190 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 200 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 210 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 220 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 230 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 240 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 250 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 260 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 270 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 280 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 290 mg of mitapivat perday. In certain embodiments, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 300 mg of mitapivat per day. In certain embodiments, mitapivat ora pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 300-360 mg of mitapivat perday. Dosing can be once, twice, or three times daily. In one aspect,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose equivalent to about 5 mg of mitapivatthereof twice per day. In one aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 10 mg of mitapivat thereof twice per day. In oneaspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 15 mg ofmitapivat thereof twice per day. In one aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 20 mg of mitapivat twiceper day. In one aspect, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about25 mg of mitapivat thereof twice per day. In one aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 30 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 35 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 40 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 45 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 50 mg of mitapivat twiceper day. In one aspect, mitapivat or a pharmaceutically acceptable saltthereof is formulated for administration at a dose equivalent to about55 mg of mitapivat thereof twice per day. In one aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 60 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 65 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 70 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 75 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 80 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 85 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 90 mg of mitapivat thereoftwice per day. In one aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 95 mg of mitapivat thereof twice per day. In one aspect, mitapivator a pharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 100 mg of mitapivat twiceper day. In another aspect, mitapivat or a pharmaceutically acceptablesalt thereof is formulated for administration at a dose equivalent toabout 5 mg of mitapivat thereof once per day. In another aspect,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose equivalent to about 10 mg of mitapivatthereof once per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 15 mg of mitapivat thereof once per day. In oneaspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 20 mg ofmitapivat once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 25 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 30 mg of mitapivat thereof once per day. In anotheraspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 35 mg ofmitapivat thereof once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 40 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 45 mg of mitapivat thereof once per day. In oneaspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 50 mg ofmitapivat once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 55 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 60 mg of mitapivat thereof once per day. In anotheraspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 65 mg ofmitapivat thereof once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 70 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 75 mg of mitapivat thereof once per day. In anotheraspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 80 mg ofmitapivat thereof once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 85 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 95 mg of mitapivat thereof once per day. In oneaspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 100 mg ofmitapivat once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 110 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 120 mg of mitapivat thereof once per day. In anotheraspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 130 mg ofmitapivat thereof once per day. In another aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 140 mg of mitapivat thereofonce per day. In another aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 150 mg of mitapivat thereof once per day. In anotheraspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 160 mg toabout 200 mg of mitapivat thereof once per day. In another aspect,mitapivat or a pharmaceutically acceptable salt thereof is formulatedfor administration at a dose equivalent to about 200 mg to about 360 mgof mitapivat thereof once per day. In one aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 5 mg of mitapivat onceevery other day. In one aspect, mitapivat or a pharmaceuticallyacceptable salt thereof is formulated for administration at a doseequivalent to about 20 mg of mitapivat once every other day. In oneaspect, mitapivat or a pharmaceutically acceptable salt thereof isformulated for administration at a dose equivalent to about 50 mg ofmitapivat once every other day. In one aspect, mitapivat or apharmaceutically acceptable salt thereof is formulated foradministration at a dose equivalent to about 100 mg of mitapivat onceevery other day. In certain embodiment, the pharmaceutically acceptablesalt of mitapivat is mitapivat sulfate.

In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof by about 70% to about 80%. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose of mitapivat or a pharmaceutically acceptable saltthereof by about 60% to about 70%. In certain embodiments, for any ofthe methods presented herein, further comprising reducing the dose ofmitapivat or a pharmaceutically acceptable salt thereof by about 50% toabout 60%. In certain embodiments, for any of the methods presentedherein, further comprising reducing the dose of mitapivat or apharmaceutically acceptable salt thereof by about 40% to about 50%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof by about 30% to about 40%. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose of mitapivat or a pharmaceutically acceptable saltthereof by about 20% to about 30%. In certain embodiments, for any ofthe methods presented herein, further comprising reducing the dose ofmitapivat or a pharmaceutically acceptable salt thereof by about 10% toabout 20%. In certain embodiments, for any of the methods presentedherein, further comprising reducing the dose of mitapivat or apharmaceutically acceptable salt thereof by about 50%. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose of mitapivat or a pharmaceutically acceptable saltthereof from the dose equivalent to about 400 mg of mitapivat to thedose equivalent to about 200 mg. In certain embodiments, for any of themethods presented herein, further comprising reducing the dose ofmitapivat or a pharmaceutically acceptable salt thereof from the doseequivalent to about 300 mg of mitapivat to the dose equivalent to about150 mg. In certain embodiments, for any of the methods presented herein,further comprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 200 mg ofmitapivat to the dose equivalent to about 100 mg. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose of mitapivat or a pharmaceutically acceptable saltthereof from the dose equivalent to about 100 mg of mitapivat to thedose equivalent to about 50 mg. In certain embodiments, for any of themethods presented herein, further comprising reducing the dose ofmitapivat or a pharmaceutically acceptable salt thereof from the doseequivalent to about 50 mg of mitapivat to the dose equivalent to about25 mg. In certain embodiments, for any of the methods presented herein,further comprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 20 mg ofmitapivat to the dose equivalent to about 10 mg. In certain embodiments,for any of the methods presented herein, further comprising reducing thedose of mitapivat or a pharmaceutically acceptable salt thereof from thedose equivalent to about 10 mg of mitapivat to the dose equivalent toabout 5 mg. In certain embodiments, for any of the methods presentedherein, further comprising reducing the dose of mitapivat or apharmaceutically acceptable salt thereof from the dose equivalent toabout 5 mg of mitapivat to the dose equivalent to about 2.5 mg.

In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 70% to about 80%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 60% to about 70%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 50% to about 60%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 40% to about 50%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 30% to about 40%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 20% to about 30%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 10% to about 20%. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose frequency of mitapivat or apharmaceutically acceptable salt thereof by about 50%. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose frequency of mitapivat or a pharmaceuticallyacceptable salt thereof from twice daily to once daily. In certainembodiments, for any of the methods presented herein, further comprisingreducing the dose frequency of mitapivat or a pharmaceuticallyacceptable salt thereof from once daily to once every other day.

In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 400 mg ofmitapivat twice daily to the dose equivalent to about 200 mg twicedaily. In certain embodiments, for any of the methods presented herein,further comprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 300 mg ofmitapivat twice daily to the dose equivalent to about 150 mg twicedaily. In certain embodiments, for any of the methods presented herein,further comprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 200 mg ofmitapivat twice daily to the dose equivalent to about 100 mg twicedaily. In certain embodiments, for any of the methods presented herein,further comprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 100 mg ofmitapivat twice daily to the dose equivalent to about 50 mg twice daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 50 mg ofmitapivat twice daily to the dose equivalent to about 25 mg twice daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 20 mg ofmitapivat twice daily to the dose equivalent to about 10 mg twice daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 10 mg ofmitapivat twice daily to the dose equivalent to about 5 mg twice daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 5 mg twicedaily of mitapivat to the dose equivalent to about 2.5 mg twice daily.

In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 400 mg ofmitapivat once daily to the dose equivalent to about 200 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 300 mg ofmitapivat once daily to the dose equivalent to about 150 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 200 mg ofmitapivat once daily to the dose equivalent to about 100 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 100 mg ofmitapivat once daily to the dose equivalent to about 50 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 50 mg ofmitapivat once daily to the dose equivalent to about 25 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 20 mg ofmitapivat once daily to the dose equivalent to about 10 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 10 mg ofmitapivat once daily to the dose equivalent to about 5 mg once daily. Incertain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 5 mg oncedaily of mitapivat to the dose equivalent to about 2.5 mg once daily.

In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 400 mg ofmitapivat twice daily to the dose equivalent to about 200 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 300 mg ofmitapivat twice daily to the dose equivalent to about 150 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 200 mg ofmitapivat twice daily to the dose equivalent to about 100 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 100 mg ofmitapivat twice daily to the dose equivalent to about 50 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 50 mg ofmitapivat twice daily to the dose equivalent to about 25 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 20 mg ofmitapivat twice daily to the dose equivalent to about 10 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 10 mg ofmitapivat twice daily to the dose equivalent to about 5 mg once daily.In certain embodiments, for any of the methods presented herein, furthercomprising reducing the dose of mitapivat or a pharmaceuticallyacceptable salt thereof from the dose equivalent to about 5 mg twicedaily of mitapivat to the dose equivalent to about 2.5 mg once daily.

In some embodiments, mitapivat or a pharmaceutically acceptable saltthereof (e.g., mitapivat sulfate) is present in the tablet compositionin an amount equivalent to about 1 to about 200 mg of mitapivat. In someembodiments, mitapivat or a pharmaceutically acceptable salt thereof(e.g., mitapivat sulfate) is present in the tablet composition in anamount equivalent to about 1 to about 150 mg of mitapivat. In someembodiments, mitapivat or a pharmaceutically acceptable salt thereof(e.g., mitapivat sulfate) is present in the tablet composition in anamount equivalent to about 1 to about 100 mg of mitapivat. In someembodiments, mitapivat or a pharmaceutically acceptable salt thereof(e.g., mitapivat sulfate) is present in the tablet composition in anamount equivalent to about 5 mg of mitapivat. In some embodiments,mitapivat or a pharmaceutically acceptable salt thereof (e.g., mitapivatsulfate) is present in the tablet composition in an amount equivalent toabout 20 mg of mitapivat. In some embodiments, mitapivat or apharmaceutically acceptable salt thereof (e.g., mitapivat sulfate) ispresent in the tablet composition in an amount equivalent to about 50 mgof mitapivat. In some embodiments, mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) is present in thetablet composition in an amount equivalent to about 75 mg of mitapivat.In some embodiments, mitapivat or a pharmaceutically acceptable saltthereof (e.g., mitapivat sulfate) is present in a tablet composition inan amount equivalent to about 100 mg of mitapivat. In certainembodiment, the pharmaceutically acceptable salt of mitapivat ismitapivat sulfate.

As used herein, the dose amount of mitapivat or a pharmaceuticallyacceptable salt thereof (e.g., mitapivat sulfate) is based on theequivalence to the free-base form of mitapivat. For example, “mitapivator pharmaceutically acceptable salt thereof present in the compositionin an amount equivalent to about 2.0 mg of mitapivat” means about 2.0 mgof free-base mitapivat or about 2.33 mg of mitapivat sulfate having thestructural formula as depicted in the background of the presentdisclosure.

Methods of Treatment (Mitapivat Monotherapy and Concomitant Medications)

In one aspect, the present disclosure provides methods of treating adisease, disorder or condition with mitapivat or a pharmaceuticallyacceptable salt thereof and a CYP3A4/5 inducer or a CYP3A4/5 inhibitoror a p-gp inhibitor; use of mitapivat and a CYP3A4/5 inducer or aCYP3A4/5 inhibitor or a p-gp inhibitor for the manufacture of amedicament for treating a disease, disorder or condition; and mitapivator a pharmaceutically acceptable salt thereof and a CYP3A4/5 inducer ora CYP3A4/5 inhibitor or a p-gp inhibitor for use in treating a disease,disorder or condition. The disease, disorder or condition treated ispyruvate kinase deficiency (PKD), sickle cell disease, or thalassemia asfurther discussed in the following embodiments, and any one disease,disorder or condition described in the section “Diseases, Disorders andConditions” provided in the present disclosure.

In one embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof and an effective amount of aninducer of cytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In another embodiment, the present disclosure relates to a method oftreating sickle cell disease in a subject, comprising administering tothe subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof and an effective amount of an inducer ofcytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In yet another embodiment, the present disclosure relates to a method oftreating thalassemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof and an effective amount of an inducer ofcytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5). In certain embodiments,the thalassemia is alpha thalassemia. In certain embodiments, thethalassemia is beta thalassemia.

In one embodiment, the method of any one of the preceding embodimentsfurther comprises:

-   -   (a) monitoring the subject for an effect of a drug-drug        interaction between mitapivat or a pharmaceutically acceptable        salt thereof and the CYP3A4/5 inducer; and    -   (b) in the event that an effect of the drug-drug interaction is        present, adjusting the effective amount of mitapivat or a        pharmaceutically acceptable salt thereof and/or the effective        amount of the CYP3A4/5 inducer being administered to the subject        to reduce the effect.

In some embodiments, the adjustment is to reduce the dose of mitapivator a pharmaceutically acceptable salt thereof. In some embodiments, theadjustment is to reduce the dose frequency of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theadjustment is to reduce the dose and dose frequency of mitapivat or apharmaceutically acceptable salt thereof. Details of the dose and/ordose frequency adjustment are shown in the dose and dose regime section.

In one embodiment, the method of the preceding embodiment furthercomprises:

-   -   (c) repeating (a) and (b) until there are no further detectable        symptoms of the drug-drug interaction.

Steps (a) and (b) can be repeated as many times as necessary until thereare no further detectable symptoms of the drug-drug interaction. In oneembodiment, step (a) comprises measuring and analyzing thepharmacokinetic (PK) such as AUC (i.e., area under the plasmaconcentration-time curve, such as AUC₀₋₂₄, AUC_(0-t), AUC_(0-inf),AUC_(%estrap)) and C_(max) values (i.e., the maximum (or peak) serumconcentration that a drug achieves in a specified compartment or testarea of the body after the drug has been administrated and before theadministration of a second dose). In one embodiment, step (a) comprisesmeasuring and analyzing other PK parameters such as but not limited toC_(L)/F (i.e., apparent total body clearance of the drug from plasma),V_(z)/F (i.e., apparent volume of distribution during terminal phaseafter non-intravenous administration), t_(1/2) (elimination half-life),t_(last), t_(max) (time to reach maximum (peak) plasma concentrationfollowing drug administration), etc.

A “drug-drug interaction” as used herein refers to a change inconcentration in a patient of a first drug (e.g., mitapivat) andconsequential change in the effect of the first drug on thesubject/patient resulting from administration of a second drug (e.g., aCYP3A4/5 inducer, a CYP3A4/5 inhibitor). The change in concentration andconsequential change in effect of the first drug resulting from thesecond drug typically occurs because of a change in activity orexpression level of a detoxification enzyme that acts to clear the firstdrug from the patient (such as CYP3A4/5), wherein the change in activityor expression level of the detoxification enzyme is caused by the seconddrug. An increase in activity or expression level of the detoxificationenzyme will tend to decrease the concentration of the first drug in thepatient and its consequential effect on the patient, whereas a decreasein activity or expression level of the detoxification enzyme will tendto increase the concentration of the first drug in the patient and itsconsequential effect on the patient.

An “effect of a drug-drug interaction” as used herein refers to adetectable and measurable effect, such as a detectable symptom, on thesubject/patient caused by the drug-drug interaction. Where theconcentration of the first drug is increased, such as when mitapivat isco-administered with a CYP3A4/5 inhibitor, the “effect” typically refersto undesired phenomena such as an overdose of the first drug, liverfailure, kidney failure, and the prevalence of side effects and adversereactions. Where the concentration of the first drug is decreased, suchas when mitapivat is co-administered with a CYP3A4/5 inducer, the“effect” typically refers to a decrease in the therapeutic efficacy ofthe first drug (e.g., mitapivat). Exemplary effects of a drug-druginteraction as used herein further include headache, insomnia, nausea,viral upper respiratory tract infection, arthralgia, diarrhoea, fatigue,hot flush, influenza, vomiting, cough, dizziness, oropharyngeal pain,pyrexia, back pain, dysmenorrhoea, and gastroenteritis, and dyspepsia,hypertriglyceridemia, hemolytic anemia, hemolysis, dizziness, etc.

In one embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof and an effective amount of aninhibitor of cytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In another embodiment, the present disclosure relates to a method oftreating sickle cell disease in a subject, comprising administering tothe subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof and an effective amount of an inhibitor ofcytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In yet another embodiment, the present disclosure relates to a method oftreating thalassemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof and an effective amount of an inhibitor ofcytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5) or a p-gp inhibitor. Incertain embodiments, the thalassemia is alpha thalassemia. In certainembodiments, the thalassemia is beta thalassemia.

In one embodiment, the method of any one of the suitable precedingembodiments further comprises:

-   -   (a) monitoring the subject for a symptom of a drug-drug        interaction between mitapivat or a pharmaceutically acceptable        salt thereof and the CYP3A4/5 inhibitor; and    -   (b) in the event that a symptom of the drug-drug interaction is        present, adjusting the effective amount of mitapivat or a        pharmaceutically acceptable salt thereof and/or the effective        amount of the CYP3A4/5 inhibitor being administered to the        subject to reduce the effect.

In some embodiments, the adjustment is to reduce the dose of mitapivator a pharmaceutically acceptable salt thereof. In some embodiments, theadjustment is to reduce the dose frequency of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theadjustment is to reduce the dose and dose frequency of mitapivat or apharmaceutically acceptable salt thereof. Details of the dose and/ordose frequency adjustment are shown in the dose and dose regime section.

In one embodiment, the method of the preceding embodiment furthercomprises:

-   -   (c) repeating (a) and (b) until there are no further detectable        symptoms of the drug-drug interaction.        Likewise, steps (a) and (b) can be repeated as many times as        necessary until here are no further detectable symptoms of the        drug-drug interaction. Ways of monitoring the subject for a        symptom of a drug-drug interaction between mitapivat or a        pharmaceutically acceptable salt thereof and the CYP3A4/5        inhibitor as recited in step (a) are as described above.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject the effective amount of mitapivator a pharmaceutically acceptable salt thereof and the effective amountof the CYP3A4/5 inducer (or inhibitor) sequentially.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject the effective amount of mitapivator a pharmaceutically acceptable salt thereof and the effective amountof the CYP3A4/5 inducer (or inhibitor) concurrently.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject the effective amount of mitapivator a pharmaceutically acceptable salt thereof and the effective amountof the CYP3A4/5 inducer (or inhibitor) sequentially and concurrently. Asused herein, “co-administered” or “in combination with” encompassesadministration of mitapivat or a pharmaceutically acceptable saltthereof and the CYP3A4/5 inducer (or inhibitor) sequentially andconcurrently, or at different intervals. In another embodiments, for themethod of any one of the embodiments, the CYP3A4/5 inducer is a mildCYP3A4/5 inducer. In another embodiments, for the method of any one ofthe embodiments, the CYP3A4/5 inhibitor is a mild CYP3A4/5 inhibitor. Inanother embodiments, for the methods of any one of the embodiments, theCYP3A4/5 inhibitor is a moderate CYP3A4/5 inhibitor.

In another aspect, the present disclosure provides methods of treating adisease, disorder or a condition with mitapivat in the absence of aCYP3A4/5 inducer or a CYP3A4/5 inhibitor or a p-gp inhibitor; use ofmitapivat in the absence of a CYP3A4/5 inducer or a CYP3A4/5 inhibitoror a p-gp inhibitor for the manufacture of a medicament for treatingdisease, disorder or a condition; and mitapivat in the absence of aCYP3A4/5 inducer or a CYP3A4/5 inhibitor or a p-gp inhibitor for use intreating a disease, disorder or a condition.

As used herein, “in the absence of” when referring to a secondary drugco-administered with mitapivat (e.g., a CYP3A4/5 inducer or a CYP3A4/5inhibitor or a p-gp inhibitor), refers to levels of the secondary drugin the subject/patient that are sufficiently low that such that anychange in the levels of the detoxification enzymes for mitapivat and thesecondary drug results in no detectable symptoms of a drug-druginteraction between mitapivat and the secondary drug.

In one embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of an inducer ofcytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In another embodiment, the present disclosure relates to a method oftreating sickle cell disease in a subject, comprising administering tothe subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of an inducer of cytochrome P4503A4 (CYP3A4) or 3A5 (CYP3A5).

In yet another embodiment, the present disclosure relates to a method oftreating thalassemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of an inducer of cytochrome P4503A4 (CYP3A4) or 3A5 (CYP3A5). In some embodiments, the thalassemia isalpha thalassemia. In some embodiments, the thalassemia is betathalassemia.

In one embodiment, in accordance with the method of any one of thesuitable preceding embodiments, administration of a CYP3A4/5 inducer, ifany, is terminated at a sufficient time interval prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof such that there is nodetectable symptom of a drug-drug interaction between mitapivat or apharmaceutically acceptable salt thereof and the CYP3A4/5 inducer. Incertain embodiments, the CYP3A4/5 inducer is a strong CYP3A4/5 inducer.In certain embodiments, the CYP3A4/5 inducer is a moderate CYP3A4/5inducer. In some embodiments, the CYP3A4/5 inducer is terminated atleast 30 days prior to the administration of the effective amount ofmitapivat or a pharmaceutically acceptable salt thereof. In someembodiments, the CYP3A4/5 inducer is terminated at least 28 days priorto the administration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 25 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 21 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 15 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 14 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 10 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 7 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inducer is terminated at least 5 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof

In one embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of an inhibitorof cytochrome P450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In another embodiment, the present disclosure relates to a method oftreating sickle cell disease in a subject, comprising administering tothe subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of an inhibitor of cytochromeP450 3A4 (CYP3A4) or 3A5 (CYP3A5).

In yet another embodiment, the present disclosure relates to a method oftreating thalassemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of an inhibitor of cytochromeP450 3A4 (CYP3A4) or 3A5 (CYP3A5). In certain embodiment, thethalassemia is alpha-thalassemia. In certain embodiment, the thalassemiais beta-thalassemia.

In one embodiment, in accordance with the method of any one of thesuitable preceding embodiments, administration of a CYP3A4/5 inhibitor,if any, is terminated at a sufficient time interval prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof such that there is nodetectable symptom of a drug-drug interaction between mitapivat or apharmaceutically acceptable salt thereof and the CYP3A4/5 inhibitor. Incertain embodiments, the CYP3A4/5 inhibitor is a strong CYP3A4/5inhibitor. In certain embodiments, the CYP3A4/5 inhibitor is a moderateCYP3A4/5 inhibitor. In some embodiments, the CYP3A4/5 inhibitor isterminated at least 21 days prior to the administration of the effectiveamount of mitapivat or a pharmaceutically acceptable salt thereof. Insome embodiments, the CYP3A4/5 inhibitor is terminated at least 15 daysprior to the administration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 14 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 10 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 7 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 3 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 2 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, theCYP3A4/5 inhibitor is terminated at least 1 day prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof

In one embodiment, the present disclosure relates to a method oftreating pyruvate kinase deficiency (PKD) in a subject, comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof in the absence of a p-gpinhibitor.

In another embodiment, the present disclosure relates to a method oftreating sickle cell disease in a subject, comprising administering tothe subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of a p-gp inhibitor.

In yet another embodiment, the present disclosure relates to a method oftreating thalassemia in a subject, comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof in the absence of a p-gp inhibitor. In certainembodiment, the thalassemia is alpha-thalassemia. In certain embodiment,the thalassemia is beta-thalassemia.

In one embodiment, in accordance with the method of any one of thesuitable preceding embodiments, administration of a p-gp inhibitor, ifany, is terminated at a sufficient time interval prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof such that there is nodetectable symptom of a drug-drug interaction between mitapivat or apharmaceutically acceptable salt thereof and the p-gp inhibitor. Incertain embodiments, the p-gp inhibitor is a strong p-gp inhibitor(e.g., amiodarone, azithromycin, captopril, carvedilol, clarithromycin,conivaptan, cyclosporine, diltiazem, dronedarone, erythromycin,felodipine, itraconazole, ketoconazole, lopinavir, ritonavir, quercetin,quinidine, ranolazine ticagrelor, and erapamil). In some embodiments,the p-gp inhibitor is terminated at least 21 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, the p-gpinhibitor is terminated at least 15 days prior to the administration ofthe effective amount of mitapivat or a pharmaceutically acceptable saltthereof. In some embodiments, the p-gp inhibitor is terminated at least14 days prior to the administration of the effective amount of mitapivator a pharmaceutically acceptable salt thereof. In some embodiments, thep-gp inhibitor is terminated at least 10 days prior to theadministration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof. In some embodiments, the p-gpinhibitor is terminated at least 7 days prior to the administration ofthe effective amount of mitapivat or a pharmaceutically acceptable saltthereof. In some embodiments, the p-gp inhibitor is terminated at least5 days prior to the administration of the effective amount of mitapivator a pharmaceutically acceptable salt thereof. In some embodiments, thep-gp inhibitor is terminated at least 3 days prior to the administrationof the effective amount of mitapivat or a pharmaceutically acceptablesalt thereof. In some embodiments, the p-gp inhibitor is terminated atleast 2 days prior to the administration of the effective amount ofmitapivat or a pharmaceutically acceptable salt thereof. In someembodiments, the p-gp inhibitor is terminated at least 1 day prior tothe administration of the effective amount of mitapivat or apharmaceutically acceptable salt thereof

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 1 mg to about 2500 mg, orabout 5 mg to 2500 mg, or about 30 mg to 2500 mg, or about 5 mg to about700 mg, or about 15 mg to about 700 mg, about 5 mg to about 360 mg,about 5 mg to about 300 mg, about 50 mg to about 300 mg, about 120 mg toabout 360 mg, about 120 mg to about 300 mg, about 5 mg to about 120 mg,about 5 mg, about 20 mg, about 50 mg, about 100 mg, about 120 mg , about200 mg, about 300 mg, or about 360 mg of mitapivat or a pharmaceuticallyacceptable salt thereof

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 1 mg to about 2500 mg ofmitapivat or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 5 mg to 2500 mg ofmitapivat or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 5 mg to about 360 mg ofmitapivat or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 5 mg to about 300 mg ofmitapivat or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 50 mg to about 300 mg ofmitapivat or a pharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 5 mg of mitapivat or apharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 20 mg of mitapivat or apharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 50 mg of mitapivat or apharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject about 100 mg of mitapivat or apharmaceutically acceptable salt thereof.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject the effective amount of mitapivator a pharmaceutically acceptable salt thereof once daily.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject the effective amount of mitapivator a pharmaceutically acceptable salt thereof twice daily.

In one embodiment, the method of any one of the preceding embodimentscomprises administering to the subject a sulfate salt of mitapivat, suchas a sulfate salt of mitapivat having the structural formula depicted inthe background of the present disclosure.

In one embodiment, in the method of any one of the suitable precedingembodiments, the CYP3A4/5 inducer is a strong inducer of CYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the CYP3A4/5 inducer is a moderate inducer of CYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the CYP3A4/5 inducer is a mild inducer of CYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inducer of CYP3A4/5 is a strong CYP3A4/5 inducerselected from a group consisting of apalutamide, enzalutamide, mitotane,phenytoin, efavirenz, nevirapine, carbamazepine, glucocorticoids,modafinil, oxcarbazepine, phenobarbital, henytoin, pioglitazone,rifabutin, rifampin, St. John's Wort, and troglitazone.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inducer of CYP3A4/5 is a moderate CYP3A4/5 inducerselected from a group consisting of bosentan, etravirine, phenobarbital,and primidone.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inducer of CYP3A4/5 is a mild CYP3A4/5 inducer selectedfrom a group consisting of armodafinil, modafinil, and rufinamide.

In one embodiment, in the method of any one of the suitable precedingembodiments, the CYP3A4/5 inducer is selected from the group consistingof aminoglutethimide, bexarotene, bosentan, carbamazepine,dexamethasone, efavirenz, fosphenytoin, griseofulvin, modafinil,nafcillin, nevirapine, oxcarbazepine, phenobarbital, phenytoin,primidone, rifabutin, rifampin, rifapentine, St. John's wort, avasimibe,dabrafenib, mifepristone, etravirine, armodafninil, glycerolphenylbutyrate, asunaprevir, rifunamide, oritavancin, clobazam,felbamate, eslicarbazepine acetate, and genistein.

In one embodiment, in the method of any one of the suitable precedingembodiments, the CYP3A4/5 inducer is rifampin. In one aspect, rifampinis administered to the subject in the form of a capsule composition. Inone aspect, about 0.1-1000 mg of rifampin is administered to thesubject. In one aspect, about 100-1000 mg of rifampin is administered tothe subject.

In one embodiment, the method of any one of the suitable precedingembodiments comprises administering about 600 mg of rifampin to thesubject. In one aspect, the method of any one of the suitable precedingembodiments comprises administering about 600 mg of rifampin to thesubject once daily.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a strong inhibitor ofCYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a moderate inhibitor ofCYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a mild inhibitor of CYP3A4/5.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is selected from the groupconsisting of toamiodarone, amprenavir, aprepitant, atazanavir,chloramphenicol, clarithromycin, conivaptan, cyclosporine, darunavir,dasatinib, delavirdine, diltiazem, erythromycin, fluconazole,fluoxetine, fluvoxamine, fosamprenavir, imatinib, indinavir, isoniazid,itraconazole, ketoconazole, lapatinib, miconazole, nefazodone,nelfinavir, posaconazole, ritonavir, quinupristin, saquinavir,tamoxifen, telithromycin, troleandomycin, verapamil, voriconazole,grapefruit, Seville oranges, limes, pomelos, fruit juices, andvegetables from mustard green family.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a strong CYP3A4/5 inhibitorselected from a group consisting of boceprevir, cobicistat, danoprevir,elvitegravir, ritonavir, grapefruit juice, indinavir, itraconazole,ketoconazole, lopinavir, paritaprevir, ombitasvir, dasabuvir,posaconazole, saquinavir, telaprevir, tipranavir, telithromycin,troleandomycin, nelfinavir, clarithromycin, nefazodone, saquinavir,suboxone, and voriconazole.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a moderate CYP3A4/5 inhibitorselected from a group consisting of aprepitant, ciprofloxacin,conivaptan, crizotinib, cyclosporine, diltiazem, dronedarone,erythromycin, fluconazole, fluvoxamine, imatinib, tofisopam, andverapamil.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is a mild CYP3A4/5 inhibitorselected from a group consisting of chlorzoxazone, cilostazol,cimetidine, clotrimazole, fosaprepitant, istradefylline, ivacaftor,lomitapide, ranitidine, ranolazine, ticagrelor.

As used herein, the terms “weak inhibitor” and ‘mild inhibitor” are usedinterchangeably.

As used herein, the terms “weak inducer” and ‘mild inducer” are usedinterchangeably.

In one embodiment, in the method of any one of the suitable precedingembodiments, the inhibitor of CYP3A4/5 is itraconazole. In one aspect,about 0.1-1000 mg of itraconazole is administered to the subject. In oneaspect, itraconazole is administered in the form of an oral solution. Inone aspect, about 50-500 mg of itraconazole is administered to thesubject.

In one embodiment, the method of any one of the suitable precedingembodiments comprises administering about 200 mg of itraconazole to thesubject. In one aspect, about 0.1-1000 mg of itraconazole isadministered to the subject. In one aspect, about 50-500 mg ofitraconazole is administered to the subject. In one aspect, about 200 mgof itraconazole is administered to the subject. In one aspect, about 200mg of itraconazole is administered to the subject once daily. In oneaspect, about 100 mg of itraconazole is administered to the subject. Inone aspect, about 100 mg of itraconazole is administered to the subjectonce daily. In one aspect, itraconazole is administered in the form ofan oral solution.

Diseases, Disorders and Conditions

Pyruvate kinase deficiency (PKD) is a glycolytic enzymopathy thatresults in life-long hemolytic anemia. In certain embodiments, thesubject having PKD is a patient having at least 2 mutant alleles in PKLRgene. In certain embodiments, the subject having PKD is a patient havingat least 2 mutant alleles in PKLR gene and at least one is a missensemutation. See Canu et al., Blood Cells, Molecules and Diseases 2016, 57,pp. 100-109. In certain embodiments, a subject having PKD has an Hbconcentration less than or equal to 10.0 g/dL. In certain embodiments,the subject having PKD is an adult not under regular transfusion (e.g.,having had no more than 4 transfusion episodes in the 12-month period upto the treatment). In certain embodiments, the subject having PKD is anadult transfusion independent (e.g., having no more than 3 units of RBCstransfused in the 12-month period prior to the treatment). In certainembodiments, the subject having PKD is an adult under regulartransfusion (e.g., having had at least 4 transfusion episodes (e.g., atleast 6 transfusion episodes) in the 12-month period prior to thetreatment). In certain embodiments, the subject having PKD has a totalnumber of at least 5 transfusion episodes during the subject's lifetime.In certain embodiments, the subject having PKD has a total number of atleast 10 transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 15transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 20transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 25transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 30transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 40transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 50transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 60transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD has a total number of at least 70transfusion episodes during the subject's lifetime. In certainembodiments, the subject having PKD is not homozygous for the R479Hmutation or does not have 2 non-missense mutations in the PKLR gene. Incertain embodiments, the subject having PKD, under regular transfusion,has hemoglobin (Hb) ≤12.0 g/dL (if male) or ≤11.0 g/dL (if female),prior to the treatment. In certain embodiments, the subject having PKD,under regular transfusion, has transfusion occurring on average lessthan or equal to once every three weeks. In certain embodiments, thesubject having PKD has received at least 0.8 mg (e.g., at least 1.0 mg)folic acid daily (e.g., for at least 21 days) prior to the treatment. Incertain embodiments, the subject with PKD under regular transfusionachieves a reduction in transfusion burden (e.g., at least 33% reductionin the number of RBC units transfused) during the 5 weeks, 10 weeks, 15weeks, 20 weeks, or 24 weeks, 28 weeks, or 32 weeks of treatment. Incertain embodiments, the subject having PKD, not under regulartransfusion (having had no more than 4 transfusion episodes in the12-month period prior to the treatment and/or no transfusion in the 3months prior to the treatment), has hemoglobin (Hb) ≤10.0 g/dLregardless of gender prior to the treatment. In certain embodiments, thesubject having PKD has undergone splenectomy. In certain embodiment, thesubject having PKD is a child 18 years old or younger. In certainembodiments, the child is transfusion dependent.

Exemplified conditions related to PKD include, but are not limited to,anemias, cholecystolithiasis, gallstones, tachycardia, hemochromatosis,icteric sclera, splenomegaly, leg ulcers, jaundice, fatigue, andshortness of breath. As described herein, PKD is a deficiency of PKR. Incertain embodiments, the deficiency of PKR is associated with a PKRmutation.

In certain embodiments, the subject with PKD achieves a hemoglobinresponse of at least 1.0 g/dL increase in Hb concentration after thetreatment compared to the baseline of prior to the treatment. In certainembodiments, the subject with PKD achieves a hemoglobin response of atleast 1.5 g/dL increase in Hb concentration from baseline prior to thetreatment. In certain embodiments, the subject with PKD achieves ahemoglobin response of at least 2.0 g/dL increase in Hb concentrationfrom baseline prior to the treatment.

In an embodiment, the mutant PKR is selected from the group consistingof A31V, A36G, G37Q, R4OW, R40Q, L73P, S80P, P82H, R86P, 190N, T93I,G95R, M107T, G111R, A115P, S120F, H121Q, S130P, S130Y, V134D, R135D,A137T, G143S, I153T, A154T, L155P, G159V, R163C, R163L, T164N, G165V,L167M, G169G, E172Q, W201R, I219T, A221Y, D221N, G222A, I224T, G232C,N253D, G263R, G263W, E266K, V269F, L272V, L272P, G275R, G275R, E277K,V280G, D281N, F287V, F287L, V288L, D293N, D293V, A295I, A295V, I310N,I314T, E315K, N316K, V320L, V320M, S330R, D331N, D331G, D331E, G332S,V335M, A336S, R337W, R337P, R337Q, D339N, D339Q, G341A, G341D, I342F,K348N, A352D, I357T, G358R, G358E, R359C, R359H, C360Y, N361D, G364D,K365M, V368F, T371I, L374P, S376I, T384M, R385W, R385K, E387G, D390N,A392T, N393D, N393S, N393K, A394S, A394D, A394V, V395L, D397V, G398A,M403I, G406R, E407K, E407G, T408P, T408A, T408I, K410E, G411S, G411A,Q421K, A423A, A423A, R426W, R426Q, E427A, E427N, A431T, R449C, I457V,G458D, A459V, V460M, A468V, A468G, A470D, T477A, R479C, R479H, S485F,R486W, R486L, R488Q, R490W, I494I, A495T, A495V, R498C, R498H, A503V,R504L, Q505E, V5061, R510Q, G511R, G511E, R518S, R531C, R532W, R532Q,E538D, G540R, D550V, V552M, G557A, R559G, R559P, N566K, M568V, R569Q,R569L, Q58X, E174X, W201X, E241X, R270X, E440X, R486X, Q501X, L508X,R510X, E538X, R559X. These mutations are described in Canu et al., BloodCells, Molecules and Diseases 2016, 57, pp. 100-109. In an embodiment,the mutant PKR is selected from G332S, G364D, T384M, K410E, R479H,R479K, R486W, R532W, R510Q, and R490W. In certain embodiments, themutant PKR is selected from A468V, A495V, 190N, T408I, and Q421K, andR498H. In certain embodiments, the mutant PKR is R532W, K410E, or R510Q.In certain embodiments, the mutant PKR is R510Q, R486W, or R479H.

In other aspects, provided are methods of treating a disease selectedfrom hemolytic anemia, hereditary spherocytosis, hereditaryelliptocytosis, abetalipoproteinemia, Bassen-Kornzweig syndrome, andparoxysmal nocturnal hemoglobinuria in a subject in need thereof,comprising administering to the subject an effective amount of mitapivator a pharmaceutically acceptable salt thereof, in combination with or inthe absence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor). Also provided is mitapivat or apharmaceutically acceptable salt thereof, in combination with or in theabsence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor), for use in treating disease selectedfrom hemolytic anemia, hereditary spherocytosis, hereditaryelliptocytosis, abetalipoproteinemia, Bassen-Kornzweig syndrome, andparoxysmal nocturnal hemoglobinuria in a subject. Further provided isthe use of mitapivat or a pharmaceutically acceptable salt thereof, incombination with or in the absence of an effective amount of a secondarydrug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor), in themanufacture of a medicament for treating a disease selected fromhemolytic anemia, hereditary spherocytosis, hereditary elliptocytosis,abetalipoproteinemia, Bassen-Kornzweig syndrome, and paroxysmalnocturnal hemoglobinuria in a subject in need thereof. In one aspect,the disease to be treated is hemolytic anemia.

Thalassemia is an inherited blood disorder in which the normal ratio ofα- to β-globin production is disrupted due to a disease-causing variantin 1 or more of the globin genes. In certain embodiments, Alpha-globinaggregates (as found in (β-thalassemia) readily precipitate, whichdisrupts the red blood cell (RBC) membrane and results in oxidativestress. In certain embodiments, Beta-globin tetramers (Hb H, found inα-thalassemia) are generally more soluble, but are still unstable andcan form precipitates. The imbalance of the globin chain synthesis canlead to a net reduction in Hb concentrations and has dramatic effects onthe survival of RBC precursors, ultimately resulting in their prematuredestruction in the bone marrow and in extramedullary sites (Cappelliniet al., 2014). In certain embodiments, the disorder results in largenumbers of red blood cells being destroyed, which leads to anemia. Incertain embodiments, the thalassemia is alpha thalassemia. In certainembodiments, the thalassemia is beta thalassemia. In other embodiments,the thalassemia is non-transfusion-dependent thalassemia. In otherembodiments, the thalassemia is beta thalassemia intermedia. In otherembodiments, the thalassemia is Hb E beta thalassemia. In otherembodiments, the thalassemia is beta thalassemia with mutations of 1 ormore alfa genes.

In certain embodiments, the subject is an adult subject withthalassemia. In certain embodiments, the subject has thalassemia such asβ-thalassemia intermedia, Hb E β-thalassemia, α-thalassemia (Hb Hdisease), or β-thalassemia with mutations of 1 or more a genes. Incertain embodiments, the subject has non-transfusion-dependentthalassemia. In certain embodiments, the subject hasnon-transfusion-dependent alpha thalassemia. In certain embodiments, thesubject has beta-thalassemia or non-transfusion-dependentbeta-thalassemia. In certain embodiments, the subject is an adult malesubject with thalassemia such as beta-thalassemia ornon-transfusion-dependent thalassemia. In certain embodiments, thesubject is a female subject with thalassemia such as beta-thalassemia ornon-transfusion-dependent thalassemia. In certain embodiments, thesubject is an adult female subject with thalassemia such asbeta-thalassemia or non-transfusion-dependent thalassemia. In certainembodiments, the subject has a hemoglobin concentration of less than orequal to 6.0 g/dL. In certain embodiments, the subject has a hemoglobinconcentration of less than or equal to 7.0 g/dL. In certain embodiments,the subject has a hemoglobin concentration of less than or equal to 8.0g/dL. In certain embodiments, the subject has a hemoglobin concentrationof less than or equal to 9.0 g/dL. In certain aspects, the subjecthaving non-transfusion-dependent thalassemia does not have a knownhistory (e.g., has been diagnosed in the past) of Hb S or Hb C forms ofthalassemia. In certain embodiments, the term “non-transfusiondependent” thalassemia refers to subjects with thalassemia having nomore than 4 (e.g., five) units of RBCs transfused during a 24-weekperiod up to the first day of administration of mitapivat and/or no RBCtransfusions in the 8 weeks prior to the first day of administration ofmitapivat. In certain embodiment, the subject is an adult over 18 yearsold. In certain embodiment, the subject is a child 18 years old oryounger.

As used herein, sickle cell disease (SCD), Hemoglobin SS disease, andsickle cell anemia are used interchangeably. Sickle cell disease (SCD)is an inherited blood disorder caused by the presence of sicklehemoglobin (HbS). In certain embodiments, subjects with SCD haveabnormal hemoglobin, called hemoglobin S or sickle hemoglobin, in theirred blood cells. In certain embodiments, people having SCD have at leastone abnormal gene causing the body to make hemoglobin S. In certainembodiments, people having SCD have two hemoglobin S genes, HemoglobinSS.

In other aspects, provided herein are methods for treating anemia in asubject in need thereof comprising administering to the subject aneffective amount of mitapivat or a pharmaceutically acceptable saltthereof, in combination with or in the absence of an effective amount ofa secondary drug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor).Also provided is mitapivat or a pharmaceutically acceptable saltthereof, in combination with or in the absence of an effective amount ofa secondary drug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor), foruse in treating anemia in a subject in need thereof. Further provided isthe use of mitapivat or a pharmaceutically acceptable salt thereof, incombination with or in the absence of an effective amount of a secondarydrug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor), in themanufacture of a medicament for treating anemia. In one aspect, theanemia to be treated is dyserythropoietic anemia.

In certain embodiments, the anemia is a dyserythropoietic anemia such ascongenital dyserythropoietic anemia type I, II, III, or IV. In certainembodiments, the anemia is hemolytic anemia. In certain embodiments, thehemolytic anemia is a congenital and/or hereditary form of hemolyticanemia such as PKD, sickle cell disease, thalassemias (e.g., alpha orbeta or non-transfusion-dependent thalassemia), hereditaryspherocytosis, hereditary elliptocytosis), paroxysmal nocturnalhemoglobinuria, abeta-liproteinemia (Bassen-Kornzweig syndrome). Incertain embodiments, the hemolytic anemia is acquired hemolytic anemiasuch as autoimmune hemolytic anemia, drug-induced hemolytic anemia. Incertain embodiments, the hemolytic anemia is anemia as part of amulti-system disease, such as the anemia of Congenital ErythropoieticPurpura, Fanconi, Diamond-Blackfan.

As used herein, the term “anemia” refers to a deficiency of red bloodcells (RBCs) and/or hemoglobin. As used herein, anemia includes alltypes of clinical anemia, for example (but not limited to): microcyticanemia, iron deficiency anemia, hemoglobinopathies, heme synthesisdefect, globin synthesis defect, sideroblastic defect, normocyticanemia, anemia of chronic disease, aplastic anemia, hemolytic anemia,macrocytic anemia, megaloblastic anemia, pernicious anemia, dimorphicanemia, anemia of prematurity, Fanconi anemia, hereditary spherocytosis,sickle cell disease, warm autoimmune hemolytic anemia, cold agglutininhemolytic anemia, osteopetrosis, thalassemia, and myelodysplasticsyndrome.

In certain embodiments, anemia can be diagnosed on a complete bloodcount. In certain embodiments, anemia can be diagnosed based on themeasurement of one or more markers of hemolysis (e.g., RBC count,hemoglobin, reticulocytes, schistocytes, lactate Dehydrogenase (LDH),haptoglobin, bilirubin, and ferritin) and/or hemosiderinuria meancorpuscular volume (MCV) and/or red cell distribution width (RDW). Inthe context of the present invention, anemia is present if an individualhas a hemoglobin (Hb) less than the desired level, for example, the Hbconcentration of less than 14 g/dL, more preferably of less than 13g/dL, more preferably of less than 12 g/dL, more preferably of less than11 g/dL, or most preferably of less than 10 g/dL.

In certain embodiments, provided herein is a method of increasing theamount of hemoglobin in a subject by administering an effective amountof mitapivat or a pharmaceutically acceptable salt thereof, incombination with or in the absence of an effective amount of a secondarydrug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor). In certainembodiments, also provided herein is a method of increasing the amountof hemoglobin in a subject having thalassemia comprising administeringto the subject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof, in combination with or in the absence of aneffective amount of a secondary drug (e.g., a CYP3A4/5 inducer or aCYP3A4/5 inhibitor). Further provided is a method of increasing theamount of hemoglobin in subjects having non-transfusion-dependentthalassemia comprising administering an effective amount of mitapivat ora pharmaceutically acceptable salt thereof, in combination with or inthe absence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor) to the subject. In certain embodiments,the provided methods increase hemoglobin concentration in the subject.In certain embodiments, the provided methods increase Hb concentrationto a desired level, for example, above 10 g/dL, more preferably above 11g/dL, more preferably above 12 g/dL, more preferably above 13 g/dL, ormost preferably above 14 g/dL. In certain embodiments, the providedmethods increase Hb concentration by at least about 0.5 g/dL. In certainembodiments, the provided methods increase Hb concentration by at leastabout 1.0 g/dL. In certain embodiments, the provided methods increase Hbconcentration by at least about 1.5 g/dL. In certain embodiments, theprovided methods increase Hb concentration by at least about 2.0 g/dL.In certain embodiments, the provided methods increase Hb concentrationby at least about 2.5 g/dL. In certain embodiments, the provided methodsincrease Hb concentration by at least about 3.0 g/dL. In certainembodiments, the provided methods increase Hb concentration by at leastabout 3.5 g/dL. In certain embodiments, the provided methods increase Hbconcentration by at least about 4.0 g/dL. In certain embodiments, theprovided methods increase Hb concentration by at least about 4.5 g/dL.In certain embodiments, the provided methods increase Hb concentrationby at least about 5.0 g/dL. In certain embodiments, the provided methodsincrease Hb concentration by at least about 5.5 g/dL. In certainembodiments, the provided methods increase Hb concentration by at leastabout 6.0 g/dL. In certain embodiments, the increase in Hb concentrationis determined from baseline at one or more assessment between week 1 andweek 20 (e.g., between week 2 and week 15, between week 3 and week 15,and between week 4 and week 12) of treatment with an effective amount ofmitapivat or a pharmaceutically acceptable salt thereof, in combinationwith or in the absence of an effective amount of a secondary drug (e.g.,a CYP3A4/5 inducer or a CYP3A4/5 inhibitor). In certain embodiments, theprovided methods increase Hb concentration as described above in femalesubjects having thalassemia (e.g., beta-thalassemia ornon-transfusion-dependent thalassemia). In certain embodiments, theprovided methods increase Hb concentration from baseline to about 12g/dL in female subjects having thalassemia (e.g., beta-thalassemia ornon-transfusion-dependent thalassemia). In certain embodiments, theprovided methods increase Hb concentration as described above in malesubjects having thalassemia (e.g., beta-thalassemia ornon-transfusion-dependent thalassemia). In certain embodiments, theprovided methods increase Hb concentration from baseline to about 13g/dL in male subjects having thalassemia (e.g., beta-thalassemia ornon-transfusion-dependent thalassemia).

In some aspects, provided herein are methods for treating hemolyticanemia in a subject in need thereof comprising administering to thesubject an effective amount of mitapivat or a pharmaceuticallyacceptable salt thereof, in combination with or in the absence of aneffective amount of a secondary drug (e.g., a CYP3A4/5 inducer or aCYP3A4/5 inhibitor). Also provided is mitapivat or a pharmaceuticallyacceptable salt thereof, in combination with or in the absence of aneffective amount of a secondary drug (e.g., a CYP3A4/5 inducer or aCYP3A4/5 inhibitor), for use in treating hemolytic anemia in a subjectin need thereof. Further provided is the use of mitapivat or apharmaceutically acceptable salt thereof, in combination with or in theabsence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor), in the manufacture of a medicament fortreating hemolytic anemia. In one aspect, the hemolytic anemia to betreated is hereditary and/or congenital hemolytic anemia, acquiredhemolytic anemia, or anemia as part of a multi-system disease.

In some aspects, provided herein are methods for activating wild-type ormutant PKR in red blood cells in a subject in need thereof comprisingadministering to the subject an effective amount of mitapivat or apharmaceutically acceptable salt thereof, in combination with or in theabsence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor). Also provided is mitapivat or apharmaceutically acceptable salt thereof, in combination with or in theabsence of an effective amount of a secondary drug (e.g., a CYP3A4/5inducer or a CYP3A4/5 inhibitor), for use in activating wild-type ormutant PKR in red blood cells in a subject in need thereof. Furtherprovided is the use of mitapivat or a pharmaceutically acceptable saltthereof, in combination with or in the absence of an effective amount ofa secondary drug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor), inthe manufacture of a medicament for activating wild-type or mutant PKRin red blood cells.

Mitapivat is an activator of PKR mutants having lower activitiescompared to the wild type, thus are useful for methods of the presentdisclosure. Such mutations in PKR can affect enzyme activity (catalyticefficiency), regulatory properties (modulation by fructose bisphosphate(FBP)/ATP), and/or thermostability of the enzyme. Examples of suchmutations are described in Valentini et al., JBC 2002. Some examples ofthe mutants that are activated by the compounds described herein includeG332S, G364D, T384M, R479H, R479K, R486W, R532W, R510Q, and R490W.Without being bound by theory, in certain embodiments, mitapivat affectsthe activities of PKR mutants by activating FBP non-responsive PKRmutants, restoring thermostability to mutants with decreased stability,or restoring catalytic efficiency to impaired mutants.

In certain embodiments, mitapivat increases the affinity of PKR tophosphoenolpyruvate (PEP). In certain embodiments, mitapivat restoresthe ability of RBCs to cover PEP and ADP to pyruvate and ATP.

In certain embodiments, provided herein are methods of reducingtransfusion frequency of a subject with PKD comprising administering tothe subject mitapivat or a pharmaceutically acceptable salt thereof, incombination with or in the absence of an effective amount of a secondarydrug (e.g., a CYP3A4/5 inducer or a CYP3A4/5 inhibitor). In certainembodiments, the transfusion frequency is reduced by at least 5% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 10% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 15% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 20% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 25% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 30% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 35% in thenumber of RBC units transfused over at least 15 weeks. In certainembodiments, the transfusion frequency is reduced by at least 40% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 5% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 10% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 15% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 20% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 25% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 30% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 35% in thenumber of RBC units transfused over at least 20 weeks. In certainembodiments, the transfusion frequency is reduced by at least 40% in thenumber of RBC units transfused over at least 20 weeks.

In some aspects, provided herein are methods of evaluating the efficacyof mitapivat therapy (monotherapy or concomitant medications with aCYP3A4/5 inducer or a CYP3A4/5 inhibitor) in a subject, the methodcomprising: administering to the subject mitapivat or a pharmaceuticallyacceptable salt thereof, in combination with or in the absence of aneffective amount of a secondary drug (e.g., a CYP3A4/5 inducer or aCYP3A4/5 inhibitor); and acquiring a value for the level of mitapivat,the level of 2,3-diphosphoglycerate (2,3-DPG), the level of adenosinetriphosphate (ATP), or the activity of PKR in the subject, to therebyevaluate the efficacy of the mitapivat therapy. In some aspects, thevalue for the level is acquired by analyzing the plasma concentration ofmitapivat and optionally, the metabolite known to be formed by CYP3A4/5.In some aspects, the level of 2,3-DPG is acquired by analyzing the bloodconcentration of 2,3-DPG. In some aspects, the level of ATP is acquiredby analyzing the blood concentration of ATP. In some aspects, theactivity of PKR is acquired by analyzing the blood concentration of a¹³C-label in the blood. In some aspects, the analysis is performed bysample analysis of bodily fluid. In some aspects, the bodily fluid isblood. In some aspects, the analysis is performed by mass spectroscopy.In some aspects, the analysis is performed by LC-MS.

In some aspects, provided herein are methods of treating a subject, themethod comprising: administering to the subject an effective amount ofmitapivat or a pharmaceutically acceptable salt thereof, in combinationwith or in the absence of an effective amount of a secondary drug (e.g.,a CYP3A4/5 inducer or a CYP3A4/5 inhibitor); and acquiring a value forthe level of mitapivat and optionally its metabolite known to be formedby CYP3A4/5, the level of 2,3-diphosphoglycerate (2,3-DPG), the level ofadenosine triphosphate (ATP), or the activity of PKR in the subject, tothereby treat the subject.

EXEMPLIFICATIONS

As depicted in the Examples below, the drug-drug interactions betweenmitapivat sulfate and a CYP3A4/5 inducer (e.g., rifampin) or a CYP3A4/5inhibitor (e.g., itraconazole) are studied. Mitapivat can be synthesizedusing the procedures described in International Patent ApplicationPublication Nos. WO 2011/002817 and WO 2016/201227.

Example 1: Mitapivat Sulfate is Primarily Metabolized by CYP3A4

CYP-enzyme phenotyping using human liver microsomes and recombinant CYPenzymes revealed that mitapivat sulfate was primarily metabolized byCYP3A4/5 (>90%), with minor contributions from CYP2C9, CYP2C8, andCYP1A2. There was evidence of metabolism-dependent inhibition of CYP2C19(largely reversible) and CYP3A4 (largely irreversible). Mitapivatsulfate was found to be a substrate and inhibitor for P-gp but not forbreast cancer resistance protein (BCRP) and was found to be a potentialinducer of human CYP2B6 and CYP3A4. Mitapivat sulfate appeared to be amild inhibitor of CYP2C8, CYP2C9, xYP2C19, CYP2D6, and CYP3A4/5 enzymes(testosterone 6β-hydroxylation), bile salts export pump (BSEP), organicanion transporting polypeptide (OATP)1B1, organic anion transporter(OAT)3, and organic cation transporter (OCT)2, and ofuridine-5′-diphospho-glucuronosyltransferase (UGT) 1A3, 1A4, and 1A9.Mitapivat sulfate does not appear to be an inhibitor of multidrugresistance-associated protein (MRP)2, MRP3, OATP1B3, and OAT1. Themetabolite was found to be a mild inhibitor of CYP2C9 and CYP2C19 andwas not an inhibitor of P-gp or BCRP under tested concentrations.

Example 2: Drug-Drug Interaction (DDI) Studies of Mitapivat Sulfate anda CYP3A4 Inducer or a CYP3A4 Inhibitor

Twenty-eight (28) healthy, adult male and female (non-childbearingpotential) subjects were enrolled in the study in total; 14 subjects ineach study part (Parts 1 and 2). A minimum of 8 female subjects wereenrolled in the study (i.e., a minimum of 4 female subjects per studypart). Each subject participated in either Part 1 or Part 2, but notboth.

Part 1

On Day 1 of Treatment Period 1, a single oral dose of 20 mg mitapivatsulfate was administered. Serial blood samples for plasma assay ofmitapivat concentrations and its CYP3A4 metabolite, referred to hereinas the “Metabolite” (structure below),

were collected from predose to 120 hours following administration ofmitapivat sulfate. In Treatment Period 2, an oral dose of 200 mgitraconazole was administered once daily (QD) for 9 consecutive days(Day 1 through Day 9 of Treatment Period 2) with a single oral dose of20 mg mitapivat sulfate coadministered on Day 5. Serial blood samplesfor plasma assay of mitapivat and the Metabolite concentrations werecollected from predose to 120 hours following coadministration ofmitapivat sulfate and itraconazole on Day 5.

In Treatment Period 1, mitapivat sulfate was administered orally withapproximately 240 mL of water. In Treatment Period 2, on Days 1 to 4,itraconazole was administered alone immediately followed byapproximately 220 mL of water, and on Day 5, itraconazole wasadministered first (no water) and was immediately followed by mitapivatsulfate administration with approximately 220 mL of water. Study drugs(mitapivat sulfate and itraconazole) were administered following anovernight fast of at least 10 hours on Day 1 of Treatment Period 1(mitapivat sulfate only) and Day 5 of Treatment Period 2 (mitapivatsulfate and itraconazole), and subjects remained fasted for 4 hoursafter dosing. On all other dosing days, itraconazole was administeredfollowing a predose fast of at least 4 hours and subjects remainedfasted for at least 2 hours after dosing.

Part 2

On Day 1 of Treatment Period 1, a single oral dose of 50 mg mitapivatsulfate was administered. Serial blood samples for plasma assay ofmitapivat and the Metabolite concentrations were collected from predoseto 120 hours following administration of mitapivat sulfate. In TreatmentPeriod 2, an oral dose of 600 mg rifampin was administered QD for 12consecutive days (Day 1 through Day 12 of Treatment Period 2) with asingle oral dose of 50 mg mitapivat sulfate coadministered on Day 8.Serial blood samples for plasma assay of mitapivat sulfate and theMetabolite concentrations were collected from predose to 120 hoursfollowing coadministration of mitapivat and rifampin on Day 8.

In Part 2, study drugs were administered with approximately 240 mL ofwater on all dosing days including the coadministration of mitapivatsulfate and rifampin on Day 8 of Treatment Period 2. Mitapivat sulfateand rifampin was administered following an overnight fast of at least 10hours on Day 1 of Treatment Period 1 (mitapivat sulfate only) and Day 8of Treatment Period 2 (both mitapivat sulfate and rifampin) and subjectsremained fasted for 4 hours after dosing. On all other dosing days,rifampin was administered following a predose fast of at least 4 hoursand subjects remained fasted for at least 2 hours after dosing. Therewas a washout period of 7 days between the mitapivat sulfate dose inTreatment Period 1 and the first itraconazole (Part 1) or rifampin (Part2) dose in Treatment Period 2. All study drugs were consumed within 5minutes for both Part 1 and Part 2.

Test and Reference Products, Dose, Duration and Mode of AdministrationPart 1

Mitapivat sulfate (20 mg tablets) administered along with Itraconazole(also referred to as Sporanox®, 10 mg/mL oral solution, is considered asthe test arm in this study. Mitapivat sulfate administered alone isconsidered as the reference arm in this study. In Period 1, a singleoral dose of 20 mg mitapivat sulfate was administered orally withapproximately 240 mL of water. In Period 2, on Day 5 (coadministrationof itraconazole and mitapivat sulfate), 200 mg itraconazole (20 mL of 10mg/mL oral solution) was administered first (no water) and wasimmediately followed by 20 mg mitapivat sulfate (1×20 mg tablet)administration with approximately 220 mL of water. On all other dosingdays, itraconazole was administered alone immediately followed byapproximately 220 mL of water. Each subject received 2 doses ofmitapivat sulfate and 9 doses of itraconazole in Part 1.

Part 2

Mitapivat sulfate (50 mg tablets) administered along with Rifampin (alsoreferred to as Rifadin®, 300 mg capsules) is considered as the test armin this study. Mitapivat sulfate (50 mg tablets) administered alone isconsidered as the reference arm in this study.

In Period 1, a single oral dose of 50 mg mitapivat sulfate wasadministered orally. In Period 2, subjects were administered 600 mgrifampin (2×300 mg capsules) QD on Day 1 to Day 12 inclusive, with 50 mgmitapivat sulfate (1×50 mg tablet) coadministered on Day 8. Study drugswere administered with approximately 240 mL of water on all dosing daysincluding the coadministration of mitapivat sulfate and rifampin on Day8 of Period 2.

Each subject received 2 doses of mitapivat sulfate and 12 doses ofrifampin in Part 2.

Duration of Treatment:

The total duration of participation including the screening period foreach subject was approximately 49 to 62 days for Part 1 andapproximately 61 or 63 days in Part 2 of the study.

In each study part, Treatment Period 1 was approximately 6 days andTreatment Period 2 was approximately 12 days; a washout phase was 7 daysbetween both treatment periods in each study part.

Criteria for Evaluation of Pharmacokinetics

In Parts 1 and 2, for mitapivat and the Metabolite, blood samples wereobtained for all subjects to determine the plasma concentrations ofmitapivat, and the Metabolite, before mitapivat sulfate dosing (0 hour)on Day 1 of Period 1 (Parts 1 and 2) and Day 5 (Part 1) or Day 8 (Part2) of Period 2 at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, 48, 72, 96,and 120 hours postdose. Plasma concentrations of mitapivat and theMetabolite were determined using methods validated with respect toaccuracy, precision, linearity, sensitivity, and specificity, with alower limit of quantitation (LLOQ) of 0.5 ng/mL for both analytes. Anoncompartmental PK approach was used to analyze individual plasmamitapivat and the Metabolite concentration time data (using Phoenix®WinNonlin® Version 7.0). The following PK parameters were calculated:AUC_(0-t), AUC_(0-inf), AUC₀₋₂₄, AUC_(%extrap), Cmax, CL/F (mitapivatonly), Tlast, Tmax, t½, and Vz/F (mitapivat only).

Statistical Methods for Pharmacokinetics

The plasma concentrations of mitapivat and the Metabolite were listedand summarized by treatment and collection time for all subjects in thePK Population. Plasma concentrations were presented with the same levelof precision as received from the bioanalytical laboratory includingsample size (n), arithmetic mean (Mean), standard deviation (SD),coefficient of variation (CV %), standard error of the mean (SEM),minimum, maximum, and median. Concentrations from excluded subjects wereincluded in the plasma concentration tables but were excluded from thesummary statistics and noted as such in the tables. All BLQ values werepresented as “BLQ” in the plasma concentration table listings andfootnoted accordingly.

PK parameters were presented with a minimum of 3 significant figures. PKparameters were listed by subject, tabulated by treatment, andsummarized using descriptive statistics (n, mean, SD, CV%, SEM, minimum,median, maximum, geometric mean (Geom. Mean) and geometric meancoefficient of variation (Geom. CV%). Time-based parameters (i.e., Tmaxand t½) and treatment:reference AUC and Cmax ratios were presented to 2decimals. Excluded subjects were included in the PK parameter tablelistings but were excluded from the summary statistics and noted as suchin the tables.

To evaluate the effect of itraconazole and rifampin on mitapivat and theMetabolite (primary objective), plasma mitapivat and the Metabolite PKparameters (Cmax, AUC_(0-t), and AUC_(0-inf)) were natural log (ln)transformed before analysis. The ln-transformed value of the PKparameters was analyzed using a linear mixed effect model for each studypart separately with treatment as a fixed effect and subject as a randomeffect. The linear mixed effect model included treatment as a fixedeffect and subject as a random effect. The inferential results(least-squares means [LSMs], difference between LSMs, and 90% confidenceintervals [CIs] of the difference) were back transformed to the originalscale. Geometric LSMs, geometric mean ratios (GMRs), and 90% CIs werepresented. The GMR of the LSMs were calculated from the back-transformeddifference between the treatment LSMs. The GMRs were expressed as apercentage relative to mitapivat sulfate given alone for both Part 1 andPart 2. The 90% CIs for the ratios were derived by back-transformationof the CIs obtained for the difference between the treatment LSMs. TheCIs were expressed as a percentage relative to mitapivat sulfate givenalone for both Part 1 and Part 2.

The median differences in Tmax for evaluation of the effect ofitraconazole and rifampin on mitapivat and the Metabolite along with therange of the difference were calculated using the non-parametricWilcoxon Signed Rank test, and the p-value was presented. Note that Tmaxwas not ln-transformed for these analyses.

Pharmacokinetic Results Part 1

Mean plasma mitapivat and the Metabolite concentration-time profilesfollowing 20 mg mitapivat sulfate administered alone (Period 1) orcoadministered with multiple doses of 200 mg itraconazole (Period 2) onlinear and semi-log scales are presented in FIGS. 1 and 2 .

Mitapivat

Mitapivat plasma concentrations were detectable in all subjects at 0.5hour (the first scheduled postdose sample) in both Periods 1 and 2.Mitapivat was still detectable up to the last sample time at 120 hourspostdose in the majority of subjects in Period 1 and in all subjects inPeriod 2.

Measurable predose mitapivat concentrations were present in all but 2subjects in Period 2 (two subjects were BLQ). As used herein, BLQ refersto below the limit of quantification. These predose concentrations were<5% of the Cmax, so PK calculations were performed.

Mitapivat concentrations exhibited a characteristic multi-compartmentalpharmacokinetic profile. When coadministered with itraconazole,mitapivat concentrations rose to a higher mean peak with a delay in timeto peak by approximately 2 hours compared to when the drug wasadministered alone. The post-peak decline of the multi-compartmentalbehavior was slower after coadministration with itraconazole whencompared to mitapivat administered alone. Overall, higher mitapivatconcentrations were experienced for a longer time period when mitapivatwas coadministered with itraconazole.

The Metabolite

When mitapivat was administered alone, the Metabolite plasmaconcentrations were detectable in all but 1 subject at 0.5 hour. TheMetabolite remained detectable all but 1 subject up to 48 hourspostdose, with quantifiable the Metabolite out to 72 hours in 3 subjectsand 96 hours in 1 subject. In Period 2 (coadministration withitraconazole), the Metabolite concentrations were delayed relative toadministration of mitapivat alone. Concentrations were not measurable inall subjects until 1.5 hours postdose. Maximum concentrations were alsoreduced. However, the Metabolite residence was prolonged in the presenceof itraconazole, with concentrations still detectable in all but 1subject throughout the 120-hour sampling interval (in 1 subject the lastmeasurable concentration was at 96 hours postdose).

The statistical comparisons of mitapivat PK parameters following 20 mgmitapivat sulfate administered alone (Period 1) or coadministered withmultiple doses of 200 mg itraconazole (Period 2) PK parameters aresummarized below in Table 1.

TABLE 1 Summary of Statistical Comparisons of Plasma MitapivatPharmacokinetic Parameters Following 20 mg Mitapivat SulfateCo-Administered With Multiple Doses of 200 mg Itraconazole VersusAdministered Alone (Part 1) Mitapivat Sulfate + Itraconazole (Test)Mitapivat sulfate alone (Reference) Parameter (unit) Geometric LSM nGeometric LSM n GMR 90% Confidence Interval Intra-subject CV % AUC_(0-t)(ng*hr/mL) 8860 14 1885 14 4.6998 4.2519-5.1949 15.05 AUC_(0-inf)(ng*hr/mL) 9475 14 1942 14 4.8780 4.4154-5.3890 14.97 C_(max) (ng/ml) 704.3 14  412.5 14 1.7074 1.5815-1.8433 11.48 Mitapivat sulfate alone:20 mg mitapivat sulfate (1 × 20 mg tablet) at Hour 0 on Day 1 (Period 1)Mitapivat sulfate + itraconazole: 200 mg itraconazole (20 mL of 10 mg/mLoral solution) QD on Days 1 to Day 9 inclusive (within ± 1 hour ofdosing time on Day 1) with 20 mg mitapivat sulfate (1 × 20 mg tablet)coadministered at Hour 0 on Day 5 (Period 2) Geometric least-squaresmeans (LSMs) are calculated by exponentiating the LSMs from the linearmixed effect model. Geometric Mean Ratio (GMR) = test/referenceIntra-subject CV % = 100 × (square root (exp[MSE]-1), where MSE =Residual variance from linear mixed effect model.

The coadministration of 200 mg itraconazole with 20 mg mitapivat sulfateincreased mitapivat exposure relative to when mitapivat sulfate wasadministered alone. The geometric mean AUC_(0-t) and AUC_(0-inf)infratios of mitapivat in the presence and absence of itraconazole were4.7 and 4.9, respectively. The geometric mean Cmax ratio of mitapivat inthe presence and absence of itraconazole was 1.7. The intra-subjectvariability was low at approximately 15% for AUCs and 11% for Cmax.

The statistical comparisons of the Metabolite PK parameters following 20mg mitapivat sulfate administered alone (Period 1) or coadministeredwith multiple doses of 200 mg itraconazole (Period 2) PK parameters aresummarized below in Table 2.

TABLE 2 Summary of Statistical Comparisons of Plasma MetabolitePharmacokinetic Parameters Following 20 mg Mitapivat SulfateCo-Administered With Multiple Doses of 200 mg Itraconazole VersusAdministered Alone (Part 1) Mitapivat Sulfate + Itraconazole (Test)Mitapivat sulfate alone (Reference) Parameter (unit) Geometric LSM nGeometric LSM n GMR 90% Confidence Interval Intra-subject CV % AUC_(0-t)(ng*hr/mL) 300.0 14 197.1 14 1.5218 1.4268-1.6231  9.65 AUC_(0-inf)(ng*hr/mL) 338.6 14 216.2 14 1.5661 1.4784-1.6590  8.63 C_(max) (ng/mL) 9.110 14  18.79 14 0.4849 0.4441-0.5295 13.20 Mitapivat sulfate alone:20 mg mitapivat sulfate (1 × 20 mg tablet) at Hour 0 on Day 1 (Period 1)Mitapivat sulfate + itraconazole: 200 mg itraconazole (20 mL of 10 mg/mLoral solution) QD on Days 1 to Day 9 inclusive (within ± 1 hour ofdosing time on Day 1) with 20 mg mitapivat sulfate (1 × 20 mg tablet)coadministered at Hour 0 on Day 5 (Period 2) Geometric least-squaresmeans (LSMs) are calculated by exponentiating the LSMs from the linearmixed effect model. Geometric Mean Ratio (GMR) = test/referenceIntra-subject CV % = 100 × (square root (exp[MSE]-1), where MSE =Residual variance from linear mixed effect model.

The coadministration of 200 mg itraconazole with 20 mg mitapivat sulfateincreased total Metabolite exposure relative to when mitapivat sulfatewas administered alone. The geometric mean AUC_(0-t) and AUC_(0-inf)ratios of the Metabolite in the presence and absence of itraconazolewere 1.5 and 1.6, respectively. The geometric mean Cmax ratio of theMetabolite in the presence and absence of itraconazole was 0.5. Theintra-subject variability was low at approximately 9% to 10% for AUCsand 13% for Cmax.

Part 2

Mean plasma mitapivat and metabolite the Metabolite concentration-timeprofiles following 50 mg mitapivat sulfate administered alone (Period 1)or coadministered with multiple doses of 600 mg rifampin (Period 2) onlinear and semi-log scales are presented in FIGS. 3 and 4 .

Mitapivat

Mitapivat plasma concentrations were detectable in all subjects at 0.5hour (the 1st scheduled postdose sample) when given alone or withrifampin. Mitapivat was still detectable up to the last sample time at120 hours postdose in all but 1 subject when given alone had a BLQconcentration at 120 hours). When coadministered with rifampin,detectable mitapivat concentrations were not present in any samplecollections from any subject beyond 24 hours postdose (except 1 subjectwith detectable mitapivat at 48 hours).

Mitapivat concentrations increased to a lower and slightly earlier peakwhen coadministered with rifampin compared to when administered alone.The peak arithmetic mean mitapivat concentrations were observed at anominal time of approximately 1 hour postdose for Period 1 and 0.5 hourpostdose for Period 2. The mean concentrations of mitapivat thendeclined in a multi exponential manner for both periods; however, thepost-peak initial decline in mitapivat concentrations was more rapid andto lower levels when coadministered with rifampin compared to dosingalone.

The Metabolite

The Metabolite plasma concentrations were detectable at 0.5 hourpostdose in all subjects after mitapivat sulfate was given alone and inall but 1 subject (1 subject was reported as BLQ) when mitapivat sulfatewas coadministered with rifampin. The Metabolite was still detectable inall but 4 subjects up to the last scheduled sampling time of 120 hourspostdose when mitapivat sulfate was given alone. When mitapivat sulfatewas coadministered with rifampin, the Metabolite remained detectable inall subjects up to 24 hours postdose, with quantifiable Metabolite outto 48 hours in 6 subjects and 72 hours in 1 subject. No Metaboliteconcentrations were detectable in samples collected beyond 72 hours.

The peak arithmetic mean Metabolite concentrations were observed at anominal time of approximately 2 hours postdose with mitapivat sulfategiven alone and 1 hour when mitapivat sulfate was coadministered withrifampin. When mitapivat sulfate was given alone, higher meanconcentrations of the Metabolite were observed over the initial 1 hourpostdose. Metabolite concentrations were higher when mitapivat sulfatewas coadministered with rifampin beyond 1 hour. The mean concentrationsof the Metabolite then declined in a multi exponential fashion for bothtreatments. Decline in Metabolite concentrations was more rapid whenmitapivat sulfate was coadministered with rifampin.

The statistical comparisons of mitapivat PK parameters following 50 mgmitapivat sulfate administered alone (Period 1) or coadministered withmultiple doses of 600 mg rifampin (Period 2) PK parameters aresummarized below in Table 3.

TABLE 3 Summary of Statistical Comparisons of Plasma MitapivatPharmacokinetic Parameters Following 50 mg Mitapivat SulfateCo-Administered With Multiple Doses of 600 mg Rifampin VersusAdministered Alone (Part 2) Mitapivat Sulfate + Itraconazole (Test)Mitapivat sulfate alone (Reference) Parameter (unit) Geometric LSM nGeometric LSM n GMR 90% Confidence Interval Intra-subject CV % AUC_(0-t)(ng*hr/mL) 486.4 14 5565 14 0.0874 0.0719-0.1062 29.73 AUC_(0-inf)(ng*hr/mL) 496.4 14 5681 14 0.0874 0.0719-0.1062 29.84 C_(max) (ng/ml)292.4 14 1263 14 0.2316 0.1933-0.2773 27.45 Mitapivat sulfate alone: 50mg mitapivat sulfate (1 × 50 mg tablet) at Hour 0 on Day 1 (Period 1)Mitapivat sulfate + rifampin: 600 mg rifampin (2 × 300 mg capsules) QDon Day 1 to Day 12 inclusive (within ± 1 hour of dosing time on Day 1)with 50 mg mitapivat sulfate (1 × 50 mg tablet) coadministered at Hour 0on Day 8 (Period 2) Geometric least-squares means (LSMs) are calculatedby exponentiating the LSMs from the linear mixed effect model. GeometricMean Ratio (GMR) = test/reference Intra-subject CV % = 100 × (squareroot (exp[MSE]-1), where MSE = Residual variance from linear mixedeffect model.

The coadministration of 600 mg rifampin with 50 mg mitapivat sulfatedecreased mitapivat exposure relative to when mitapivat sulfate wasadministered alone. The geometric mean AUC_(0-t) and AUC_(0-inf)infratios of mitapivat in the presence and absence of rifampin were0.09. The geometric mean Cmax ratio of mitapivat in the presence andabsence of rifampin was 0.23. The intra-subject variability was low atapproximately 30% for AUCs and 27% for Cmax.

The statistical comparisons of Metabolite PK parameters following 50 mgmitapivat sulfate administered alone (Period 1) or co-administered withmultiple doses of 600 mg rifampin (Period 2) PK parameters aresummarized below in Table 4.

TABLE 4 Summary of Statistical Comparisons of Plasma MetabolitePharmacokinetic Parameters Following 50 mg Mitapivat SulfateCo-Administered With Multiple Doses of 600 mg Rifampin VersusAdministered Alone (Part 2) Mitapivat Sulfate + Itraconazole (Test)Mitapivat sulfate alone (Reference) Parameter (unit) Geometric LSM nGeometric LSM n GMR 90% Confidence Interval Intra-subject CV % AUC_(0-t)(ng*hr/mL) 230.5 14 623.0 14 0.3700 0.3316-0.4129 16.50 AUC_(0-inf)(ng*hr/mL) 244.9 14 662.6 14 0.3696 0.3321-0.4113 16.08 C_(max) (ng/mL) 64.47 14  53.80 14 1.1983 1.0642-1.3492 17.86 Mitapivat sulfate alone:50 mg mitapivat sulfate (1 × 50 mg tablet) at Hour 0 on Day 1 (Period 1)Mitapivat sulfate + rifampin: 600 mg rifampin (2 × 300 mg capsules) QDon Day 1 to Day 12 inclusive (within ± 1 hour of dosing time on Day 1)with 50 mg mitapivat sulfate (1 × 50 mg tablet) coadministered at Hour 0on Day 8 (Period 2) Geometric least-squares means (LSMs) are calculatedby exponentiating the LSMs from the linear mixed effect model. GeometricMean Ratio (GMR) = test/reference Intra-subject CV % = 100 × (squareroot (exp[MSE]-1), where MSE = Residual variance from linear mixedeffect model.

The coadministration of 600 mg rifampin with 50 mg mitapivat sulfatedecreased the Metabolite exposure relative to when mitapivat sulfate wasadministered alone. The geometric mean AUC0-t and AUC0-inf ratios of theMetabolite in the presence versus absence of rifampin were 0.37. Thegeometric mean Cmax ratio of the Metabolite in the presence versusabsence of rifampin was 1.2. The intra-subject variability was low atapproximately 16% to 18% for AUCs and Cmax.

Conclusions

Total exposure of mitapivat increased in the presence of itraconazolecompared to dosing of mitapivat sulfate alone.

Total exposure of mitapivat decreased in the presence of rifampincompared to dosing of mitapivat sulfate alone.

Example 3. Development of a PBPK Model for Mitapivat Sulfate to Evaluatethe Potential for Drug-Drug Interactions Part I

A physiologically based pharmacokinetic model (hereinafter “PBPK” model)for mitapivat sulfate based on the currently available in-vitro andclinical PK data is being developed. The developed PBPK model will beused to assess DDI liability with mitapivat sulfate acting as a victimof CYP3A4-mediated metabolism as well as a perpetrator of CYP andtransporter mediated DDI.

Specifically, a PBPK model that includes a simple first order absorptionmodel will be developed. Full PBPK and minimal PBPK distribution modelswill be evaluated, both of which consider liver and intestinalmetabolism. The fraction of mitapivat sulfate absorbed will be estimatedfrom mass balance data from the [14C] human ADME study (Clinical StudyAG348-C-009). In-vitro data will be used to assign the relativecontribution of CYP3A4 to the clearance of mitapivat sulfate. K_(I) andk_(inact) values for mechanism based inhibition of CYP3A4 and CYP3A4induction (Emax and EC50) data by mitapivat sulfate will be incorporatedwithin the PBPK model to assess the net DDI effects at steady state.Once an mitapivat sulfate PBPK model has been verified against theavailable clinical data, a series of DDI simulations assessing mitapivatsulfate as a victim or perpetrator of DDI will be performed in healthyvolunteers.

Experiments With Mitapivat Sulfate as a Victim

The impact of the co-administration of the strong CYP3A4 inhibitor,itraconazole, on the PK of mitapivat sulfate will be determined based onthe following single and multiple-dose administration of 5, 20 and 50 mgBID.

The impact of the co-administration of the strong CYP3A4 inhibitor,ketoconazole, on the PK of mitapivat sulfate will be determined basedfollowing single and multiple-dose administration of 5, 20 and 50 mgBID.

The impact of the co-administration of the moderate CYP3A4 inhibitorfluconazole on the PK of mitapivat sulfate will be determined based onthe following single-and multiple dose administration of 5, 20 and 50 mgBID.

The impact of the co-administration of the mild CYP3A4 inhibitorcimetidine on the PK of mitapivat sulfate will be determined based onthe following single- and multiple dose administration of 5, 20 and 50mg BID.

The impact of the co-administration of the strong CYP3A4 inducer,rifampin, on the PK of mitapivat sulfate will be determined based on thefollowing single- and multiple dose administration of 5, 20 and 50 mgBID.

The impact of the co-administration of the moderate CYP3A4 inducerefavirenz on the PK of mitapivat sulfate will be determined followingsingle- and multiple dose administration of 5, 20 and 50 mg BID.

Experiments With Mitapivat Sulfate as Perpetrator

The extent to which 5 to 50 mg BID mitapivat sulfate affects thesystemic exposure of CYP3A4 substrate midazolam when co-administeredwith mitapivat sulfate will be determined.

The effect of 5 to 50 mg mitapivat sulfate on the systemic exposures ofCYP2B6 substrate bupropion when co-administered will be determined.CYP2B6 induction (Emax and EC50) data by mitapivat sulfate will beincorporated within the PBPK model. Sensitivity analysis will beconducted to gauge the impact of the uncertainty around Emax and EC50 onthe predicted DDIs.

The effect of 5 to 50 mg AG mitapivat sulfate BID on the systemicexposures of CYP2C9 substrate warfarin when co-administered will bedetermined. CYP2C9 induction (Emax and EC50) data by mitapivat sulfatewill be incorporated within the PBPK model. Sensitivity analysis will beconducted to gauge the impact of the uncertainty around Emax and EC50 onthe predicted DDIs.

The effect of 5 to 50 mg mitapivat sulfate BID on the systemic exposuresof CYP2C8/CYP3A4 substrate repaglinide when co-administered will bedetermined. CYP2C8 induction (Emax and EC50) data by mitapivat sulfatewill be incorporated within the PBPK model. Sensitivity analysis will beconducted to gauge the impact of the uncertainty around Emax and EC50 onthe predicted DDIs.

The effect of 5 to 50 mg mitapivat sulfate BID on the systemic exposuresof CYP2C19 substrate omeprazole when co-administered will be determined.CYP2C19 induction (Emax and EC50) data by mitapivat sulfate will beincorporated within the PBPK model. Sensitivity analysis will beconducted to gauge the impact of the uncertainty around Emax and EC50 onthe predicted DDIs.

The effect of mitapivat sulfate (5 to 50 mg BID) on the systemicexposures of OATP1B1/OATP1B3 (rosuvastatin), P-gp (digoxin), metformin(MATE1 and OCT2), OAT3 (methotrexate) substrate drugs whenco-administered will be determined. The respective IC50 values forinhibition of OATP1B1, P-gp, MATE1, OCT2 and OAT3 by mitapivat sulfatewill be incorporated within the PBPK model. Sensitivity analysis will beperformed to assess the impact of these parameters on the DDI liabilityof mitapivat sulfate.

Part II

The first order absorption component of the previously developed PBPKmodel will be extended using available pH solubility data to include theADAM model. The recovery of mitapivat sulfate plasma concentrationprofiles after single and multiple dosing (up to 50 mg BID) to healthyvolunteer subjects will be demonstrated. Once verified against theavailable clinical data, the modified PBPK model will be used to assessthe impact of an increase in gastric pH on the pharmacokinetics ofmitapivat sulfate via manipulation of system parameters (gastric pH).

While a number of embodiments have been described, the scope of thisdisclosure is to be defined by the appended claims, and not by thespecific embodiments that have been represented by way of example. Thecontents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

1-45. (canceled)
 46. A method of treating hemolytic anemia in a subject, comprising administering to the subject about 10 mg/day, about 40 mg/day, about 100 mg/day, or about 200 mg/day of mitapivat sulfate and a moderate inducer of cytochrome P450 3A4/5 (CYP3A4/5).
 47. The method of claim 46, wherein the subject is administered about 10 mg/day of mitapivat sulfate.
 48. The method of claim 47, wherein the subject is administered about 5 mg of mitapivat sulfate twice per day.
 49. The method of claim 46, wherein the subject is administered about 40 mg/day of mitapivat sulfate.
 50. The method of claim 49, wherein the subject is administered about 20 mg of mitapivat sulfate twice per day.
 51. The method of claim 46, wherein the subject is administered about 100 mg/day of mitapivat sulfate.
 52. The method of claim 51, wherein the subject is administered about 50 mg of mitapivat sulfate twice per day.
 53. The method of claim 46, wherein the subject is administered about 200 mg/day of mitapivat sulfate.
 54. The method of claim 53, wherein the subject is administered about 100 mg of mitapivat sulfate twice per day.
 55. The method of claim 46, wherein the mitapivat sulfate is orally administered as part of a tablet composition.
 56. The method of claim 48, wherein the mitapivat sulfate is orally administered as part of a tablet composition.
 57. The method of claim 50, wherein the mitapivat sulfate is orally administered as part of a tablet composition.
 58. The method of claim 52, wherein the mitapivat sulfate is orally administered as part of a tablet composition.
 59. The method of claim 54, wherein the mitapivat sulfate is orally administered as part of a tablet composition.
 60. The method of claim 55, wherein the tablet composition further comprises microcrystalline cellulose, mannitol, croscarmellose sodium, and sodium stearyl fumarate.
 61. The method of claim 56, wherein the tablet composition further comprises microcrystalline cellulose, mannitol, croscarmellose sodium, and sodium stearyl fumarate.
 62. The method of claim 57, wherein the tablet composition further comprises microcrystalline cellulose, mannitol, croscarmellose sodium, and sodium stearyl fumarate.
 63. The method of claim 58, wherein the tablet composition further comprises microcrystalline cellulose, mannitol, croscarmellose sodium, and sodium stearyl fumarate.
 64. The method of claim 59, wherein the tablet composition further comprises microcrystalline cellulose, mannitol, croscarmellose sodium, and sodium stearyl fumarate. 